CA1064420A - Antiprotozoal agent for animals - Google Patents
Antiprotozoal agent for animalsInfo
- Publication number
- CA1064420A CA1064420A CA257,348A CA257348A CA1064420A CA 1064420 A CA1064420 A CA 1064420A CA 257348 A CA257348 A CA 257348A CA 1064420 A CA1064420 A CA 1064420A
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- Prior art keywords
- antibiotic
- salt
- cultivation
- medium
- benzene
- Prior art date
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
ABSTRACT OF THE DISCLOSURE
This invention discloses novel antiprotozoal drug for animals and a process for its manufacture. The antibiotic of the general formula I
This invention discloses novel antiprotozoal drug for animals and a process for its manufacture. The antibiotic of the general formula I
Description
~644ZO
______.________________ This invention relates generally to a new antibiotic and in particular, to a new antibiotic use~ul as an antiprotozoal drug for animals and to a pro~ss for producing the ant}biotic.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is the infrared absorption spectrum (in chloro-form) of the novel antibiotic;
Figure 2 is the nuclear magnetic resonance spectrum (in CDC13) of the novel antibiotic;
Figure 3 is the structure of the antibiotic (DE-3936)Na salt.
DETAILED DESCRIPTION OF THE INVENTION
,, _ _ _ _ _ _ _ _ , This invention relates to a new antibiotic and more ..
particularly to antibiotic DE-3936 and process for producing thereof and to an antiprotozoal drug for animals containing the antibiotic.
.
The new antibiotic, DE-3936, was isolated from the ~, ZO
fermentation broth of a streptomycetes No. 9735-1. The strain was isolated from a soil sample collected at Musashino-shi, Tokyo and identified as a strain of Streptomyces hygroscopicus.
The microbiological properties of the strain are as follows:
,. :
1) Morphological characteristics Morphological characteristics of the strain were observed with cultures incubated at 28C for 10 to 14 days on various media, such as ammonium Czapek's agar, glucose asparagine agar, inorganic salts-starch agar, tyrosine agar and glycerol starch glutamate agar. Microscopical observations of morpholo-gical characteristics were made with both optical and electron microscopes.
"~ .
.. .. : . .. . . . .
` 1064420 1 The following data were mainly observed with cultures on glucose asparagine agar: Su~strate mycelium is flexuous or wavy, and does not fragment into bacillary or coccoid forms.
Conidiophores are formed as short branches located along straight or flexuous main axial hyphae of aerial mycelia, and terminated in mostly closed spirals of two or more turns, sometimes in hook- and loop-like spirals, seldom in open spirals. Conidia are oval to short cylindrical (0.8~1.0~ x 1.0~1.5~ ), sometimes non-segmented, with warty surface. Whirls, zoospores, sporangia 0 or sclerotia were not observed.
______.________________ This invention relates generally to a new antibiotic and in particular, to a new antibiotic use~ul as an antiprotozoal drug for animals and to a pro~ss for producing the ant}biotic.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is the infrared absorption spectrum (in chloro-form) of the novel antibiotic;
Figure 2 is the nuclear magnetic resonance spectrum (in CDC13) of the novel antibiotic;
Figure 3 is the structure of the antibiotic (DE-3936)Na salt.
DETAILED DESCRIPTION OF THE INVENTION
,, _ _ _ _ _ _ _ _ , This invention relates to a new antibiotic and more ..
particularly to antibiotic DE-3936 and process for producing thereof and to an antiprotozoal drug for animals containing the antibiotic.
.
The new antibiotic, DE-3936, was isolated from the ~, ZO
fermentation broth of a streptomycetes No. 9735-1. The strain was isolated from a soil sample collected at Musashino-shi, Tokyo and identified as a strain of Streptomyces hygroscopicus.
The microbiological properties of the strain are as follows:
,. :
1) Morphological characteristics Morphological characteristics of the strain were observed with cultures incubated at 28C for 10 to 14 days on various media, such as ammonium Czapek's agar, glucose asparagine agar, inorganic salts-starch agar, tyrosine agar and glycerol starch glutamate agar. Microscopical observations of morpholo-gical characteristics were made with both optical and electron microscopes.
"~ .
.. .. : . .. . . . .
` 1064420 1 The following data were mainly observed with cultures on glucose asparagine agar: Su~strate mycelium is flexuous or wavy, and does not fragment into bacillary or coccoid forms.
Conidiophores are formed as short branches located along straight or flexuous main axial hyphae of aerial mycelia, and terminated in mostly closed spirals of two or more turns, sometimes in hook- and loop-like spirals, seldom in open spirals. Conidia are oval to short cylindrical (0.8~1.0~ x 1.0~1.5~ ), sometimes non-segmented, with warty surface. Whirls, zoospores, sporangia 0 or sclerotia were not observed.
2) Cultural characteristics All cultures were incubated at 28C for 21 days and observations were carried out every 7 days after inoculation.
The cultural characteristics are shown in Table 1.
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The cultural characteristics are shown in Table 1.
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3) Physiological characteristics The physiological characteristics, including the utilization of carbon sources, were investigated with the methods of Pridham and Gottlieb (J. Bact. 56, page 107~114 ~1948)).
~ rom the results, the characteristics of strain No.
9735-1 are summarized as follows: (1) The strain No. 9735-1 belongs to the genus Streptomyces. (2) The aerial mycelium exhibits hygroscopic properties. (3) The conidiophores terminate in mostly closed spirals. The surface of conidia is warty.
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3) Physiological characteristics The physiological characteristics, including the utilization of carbon sources, were investigated with the methods of Pridham and Gottlieb (J. Bact. 56, page 107~114 ~1948)).
~ rom the results, the characteristics of strain No.
9735-1 are summarized as follows: (1) The strain No. 9735-1 belongs to the genus Streptomyces. (2) The aerial mycelium exhibits hygroscopic properties. (3) The conidiophores terminate in mostly closed spirals. The surface of conidia is warty.
(4) The color of the aerial mycelium is brownish gray on most media, and no soluble pigment is produced on most media. (5) -Tyrosinase reaction, milk coagulation and cellulose decomposition are negative. Nitrate reduction, starch hydrolysis, gelatin lique-factl~on and milk peptonization are positive. ~6) The chromogenicity is negative ~on chromogenic type). ~7) Carbon sources are generally uti`lized well as shown in Table 2, TABLE 2 Carbon source utilization of strain No. 9735-1 Carbon source Growth Carbon source Growth ........ ~
L-Arabinose + Starch +++
D-Xylose + Inulin ++
L-Rhamnose +++ Dulcitol D-Glycose +++ Inocitol D-Galactose+++ D-Sorbitol D-Fructose ++ Glycerol +++
D-Mannose + Salicin -Sucrose +++ D-Mannitol +++
Lactose +++ Na-Citrate D-Maltose + Na-Succinate +
Trehalose + Na-Acetate Raffinose +++ Negative control . ~ _ .. .. ...... .
+++ : good growth ++ : moderate growth +~+ : poor growth - : no growth 1 (8) Optimum temperature and pH for growth are 26-30C and pH
6-7, respectively.
4) Comparison of strain No. 9735-1 with other known species Among the known species of Streptomyces described in "Bergey's Manual of Determinative sacteriology" 7 Ed. and 8 Ed., Waksman's "The Actinomycetes; Vol. II, the ISP reports; [Intern.
J. Syst. Bact. 22 page 307-309 (1972)], and the other literature, Streptomyces hygroscopicus (Jensen) Waksman and Henrici is closely related to strain No~ 9735-1. However, there were some 1~ differences in the following points: Formation of aerial mycelium on nutrient agar, soluble pigment on sucrose Czapek's agar and glucose asparagine agar, nitrate reduction and carbon source utilization. TreSner and Backus (Appln. Microbiol 4:
pages 243-250 (1956)) pointed out the following three fundamental characteristics of Streptomyces hygroscopicus: (1) The brownish- `
gray (Mouse Gray to Benzo brown of Ridgway, 19~2) color of the .! spore in the mass. (2) The tightly wound coils of the spore-bearing hyphae. (3) The characteristics black hygroscopic areas - on certain media.- Strain No. 9735-1 has such fundamental characteristics of Streptomyces hygroscopicus. Therefore, it is reasonable to conclude that strain No.9735-1 is one of the -~ strains of Streptomyces hygroscopicus (Jensen) Waksman and Henrici (1948)j and it was named Streptomyces hygroscopicus No. 9735-1. This strain has been deposited in the Fermentation Research Institute, Chiba, Japan as Ferm-P3159.
. . .
Antibiotic DE-3936 of this invention is obtained by, for example, cultivating the strain 9735-1 using a proper culture medium under aerobic conditions by the manner usually employed for the cultivation of actinomycetes such as shaking cultivation and submerged cultivation.
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`" 1064420 `~ 1 For the culture medium, any medium containing suitable nutrient sources can be used and as to carbon source, glucose, fructose, sucrose, dextrin, starch, molasses, glycerol, etc., may be used although the use of glucose is most preferable.
Furthermore, nitrogen source which can be used for the cultiva-tion includes, a soybean meal, cotton seed flour, distiller's soluble, corn steep liquor, yeast, yeast extract, polypeptone, etc., and out of them, soybean meal, cotton seed flour, and distiller's soluble are most preferable. Moreover, the culture 0 medium used in this invention may further contain a fatty acid ~-or an ester or salt thereof as well as other additives ordinary used for the cultivation of actinomycetes, such as an inorganic salt, slight amounts of other organic and inorganic components, an antifoaming agent, etc.
The pH of the initial culture medium may be in a range suitable for the growth of actinomycetes but is preferably in the range of from 6.5 to 7.5. The cultivation temperature may ~ be also in the range suitable for the growth of the strain in - ~ this invention but i5 preferably in the range of from 27C to 31C for producing effectively the substance DE-3936. The : -., accumulation of DE-3936 in the culture medium reached its maximum ;; usually after 65_110 hours.
When a comparatively small amount of substance DE-3936 is required, the cultivation may be carried out by shaking `~ cultivation but in order to produce a large amount of substance -; DE-3936 in an industrial scale, it is advantageous to employ an aerobic submerged cultivation using, for example, a fermentation -tank.
The outline of the cultivation process by tank fermen-tation is as follows: To a culture medium in a tank the spore ~' ~ - 8 -suspension of Streptomyces hygroscopicus No. 9735-1 may be directly inoculated but such a direct inoculation may after cause the delay of the growth, therefore, it is preferred to prepare the Seed culture of the strain prior to tank fermentation.
The activated seed culture is inoculated to the culture medium (about 0.2 - 2% of the seed culture is added to the culture medium) and the aerobic submerged cultivation is carried out.
The blow amount of sterile air supplied to the culture medium varies according to the type of the fermentation tank but is usually 0.2-1.0 V V M (i.e., 0.2-1.0 volume of air per volume of culture per minute) for obtaining the sufficient accumulation of DE-3936.
Substance DE-3936 can be collected from the fermentation broth as the form of free acid or a salt thereof by an ordinary manner. That is, since the free acid of DE-3936 or salts thereof is soluble in various organic solvents, the product is first extracted from the fermentation broth with a proper organic solvent and then is purified by chromatography, recrystallization, ~ etc. In this case, it is preferable that the extraction is carried out after adjusting the pH of the fermenation broth to 7.0-8.0 using, for example, an aqueous sodium hydroxide solution.
-~ However, in many cases, the pH of the broth after the fermentation is in the aforesaid range, so the control of pH may be not necessary. The extraction is carried out usually after separating - the fermentation broth into mycelial cake and filtrate broth by filtration both of which contain DE-3936. The antibiotic is - extracted, for example, with methanol or acetone from the cake and with benzene, chloroform, dichloroethane or ethyl acetate from the filt_ate broth. Then, each solvent layer containing DE-3936 is separated from the aqueous layer or the solid and these solvent layers are combined. After concentrating by _ g _ -~0644Z0 evaporation under a reduced pressure, the mixture is purified by, for example, a column chromatography containing alumina.
n this case, benzene, ethyl acetate, methanol, or a mixed solvent of them is used as the developing solvent.
The eluate is divided in to fractions and the fractions containing DE-3936 are monitored with antimicrobial activity or color reaction (vanilin-H2S04) and desirable fractions are collec~1 The fraction thus collected is concentrated under a reduced pressure and is further purified by the proper combination of a column chromatography of Cephadex* LH-20 (methanol is proper as the developed solvent), or silica gel (benzene, acetone or a mixture of them is suitable as the developing solvent), and the - treatment with activated carbon. The fractions containing DE-3936 are collected and concentrated under a reduced pressure to provide white powder of DE-3936. The powder may be crystallized by a conventional manner. DE-3936 is dissolved in a small amount of, for e~ample, acetone and after the addition of petroleum ether thereto, the mixture is allowed to stand at room temperature or ` in a cold place to provide the colorless prisms or pillars of DE-3936. The crystal obtained is a complex or composition of the salt of DE-3936, which consists of mainly sodium salt thereof and in addition potassium salt and ammonium salt. The ratio of the components in the product may vary according to the condition of the fermentation.
The antibiotic, DE-3936,thus obtained can be converted to any of the required form such as free acid, sodium salt, potassium salt, ammonium salt, silver salt, etc. by the following manner. The substance DE-3936 obtained from the fermentation broth is dissolved in an organic solvent immiscible with water, such as benzene, chloroform, etc., and the solution is shaken with diluted hydrochloric acid. This operation is repeated several * Trade Mark 1 times, the solvellt layer is then separated and concentrated to provide the white powder of the free acid of D~-3936. sy re-crystallizing the white powder thus obtained from benzene or a petroleum solvent, the pure free acid of DE-3936 is obtained as the colorless pillars or prisms.
Alternatively, the free acid can be obtained by another method. That is, the substance DE-3936 is dissolved in acetone and a proper amount of water is added to the solution, then the pH of the mixture is adjusted to 2-4 with diluted hydrochloric acid, and then the substance is extracted with a water-immiscible solvent such as benzene and the like.
DE-3936 may be also ob~ained as the sodium salt, potassium salt, or ammonium salt by the following manner. That is, the free acid of the substance DE-3936 is dissolved in acetone, to the solution a proper amount of water is added and the pH of the solution is adjusted to 9 - 12 by adding thereto an aqueous solu-tion of sodium hydroxide, potassium hydroxide, or ammonia, and after removing acetone from the mixture by concentration, ~he substance is extracted with a solvent such as benzene, ethyl acetate, chloroform, etc., followed by concentration, whereby ..
~; the corresponding salt of DE-3936 is obtained as the colorless powder. Furthermore, by recrystallizing the colorless powder thus obtained from a suitable solvent system such as benzene-petroleum ether, benzene-n-hexane, or water-methanol, the salt of DE-3936 is obtained as needles, pillars, or prisms.
Also, the silver salt of substance DE-3936 is obtained -~by dissolving the sodium salt or potassium salt of DE-3936 in methanol and then adding to the solution an aqueous solution of silver nitrate. Furthermore, by recrystallizing the silver salt of DE-3936 from a ben~ene-petroleum ether or a water-methanol, ~ ...
. -- 11 --~, '' : , ' ~
1 the silver salt of DE-3936 is obtained as the pillars or prisms.
The sodium salt of DE-3936 obtained by recrystallizing from benzene-pe-troleum ether possesses the following physical and chemical properties:
1. Appearance of the substance:
Colorless crystals in the for~ of needles, pillars or prisms, which are almost neutral as the sodium salt of the monocar-boxylic acid.
2. Melting point:
173.0 - 176.0C. (by a capillary method). Since the sodium salt melts with slightly turning to brown the salt is considered to melt with decomposition.
3. Optical rotation:
[~]25 ~ 67.0 (C = 1, chloroform).
4. Solubility: Easily soluble in lower alcohols, esters, , chloroform, carbon etrachloride, ethers, benzene, etc.;
soluble in cyclohexane, n-hexane, petroleum ether, etc.; but almost insoluble in water.
L-Arabinose + Starch +++
D-Xylose + Inulin ++
L-Rhamnose +++ Dulcitol D-Glycose +++ Inocitol D-Galactose+++ D-Sorbitol D-Fructose ++ Glycerol +++
D-Mannose + Salicin -Sucrose +++ D-Mannitol +++
Lactose +++ Na-Citrate D-Maltose + Na-Succinate +
Trehalose + Na-Acetate Raffinose +++ Negative control . ~ _ .. .. ...... .
+++ : good growth ++ : moderate growth +~+ : poor growth - : no growth 1 (8) Optimum temperature and pH for growth are 26-30C and pH
6-7, respectively.
4) Comparison of strain No. 9735-1 with other known species Among the known species of Streptomyces described in "Bergey's Manual of Determinative sacteriology" 7 Ed. and 8 Ed., Waksman's "The Actinomycetes; Vol. II, the ISP reports; [Intern.
J. Syst. Bact. 22 page 307-309 (1972)], and the other literature, Streptomyces hygroscopicus (Jensen) Waksman and Henrici is closely related to strain No~ 9735-1. However, there were some 1~ differences in the following points: Formation of aerial mycelium on nutrient agar, soluble pigment on sucrose Czapek's agar and glucose asparagine agar, nitrate reduction and carbon source utilization. TreSner and Backus (Appln. Microbiol 4:
pages 243-250 (1956)) pointed out the following three fundamental characteristics of Streptomyces hygroscopicus: (1) The brownish- `
gray (Mouse Gray to Benzo brown of Ridgway, 19~2) color of the .! spore in the mass. (2) The tightly wound coils of the spore-bearing hyphae. (3) The characteristics black hygroscopic areas - on certain media.- Strain No. 9735-1 has such fundamental characteristics of Streptomyces hygroscopicus. Therefore, it is reasonable to conclude that strain No.9735-1 is one of the -~ strains of Streptomyces hygroscopicus (Jensen) Waksman and Henrici (1948)j and it was named Streptomyces hygroscopicus No. 9735-1. This strain has been deposited in the Fermentation Research Institute, Chiba, Japan as Ferm-P3159.
. . .
Antibiotic DE-3936 of this invention is obtained by, for example, cultivating the strain 9735-1 using a proper culture medium under aerobic conditions by the manner usually employed for the cultivation of actinomycetes such as shaking cultivation and submerged cultivation.
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`" 1064420 `~ 1 For the culture medium, any medium containing suitable nutrient sources can be used and as to carbon source, glucose, fructose, sucrose, dextrin, starch, molasses, glycerol, etc., may be used although the use of glucose is most preferable.
Furthermore, nitrogen source which can be used for the cultiva-tion includes, a soybean meal, cotton seed flour, distiller's soluble, corn steep liquor, yeast, yeast extract, polypeptone, etc., and out of them, soybean meal, cotton seed flour, and distiller's soluble are most preferable. Moreover, the culture 0 medium used in this invention may further contain a fatty acid ~-or an ester or salt thereof as well as other additives ordinary used for the cultivation of actinomycetes, such as an inorganic salt, slight amounts of other organic and inorganic components, an antifoaming agent, etc.
The pH of the initial culture medium may be in a range suitable for the growth of actinomycetes but is preferably in the range of from 6.5 to 7.5. The cultivation temperature may ~ be also in the range suitable for the growth of the strain in - ~ this invention but i5 preferably in the range of from 27C to 31C for producing effectively the substance DE-3936. The : -., accumulation of DE-3936 in the culture medium reached its maximum ;; usually after 65_110 hours.
When a comparatively small amount of substance DE-3936 is required, the cultivation may be carried out by shaking `~ cultivation but in order to produce a large amount of substance -; DE-3936 in an industrial scale, it is advantageous to employ an aerobic submerged cultivation using, for example, a fermentation -tank.
The outline of the cultivation process by tank fermen-tation is as follows: To a culture medium in a tank the spore ~' ~ - 8 -suspension of Streptomyces hygroscopicus No. 9735-1 may be directly inoculated but such a direct inoculation may after cause the delay of the growth, therefore, it is preferred to prepare the Seed culture of the strain prior to tank fermentation.
The activated seed culture is inoculated to the culture medium (about 0.2 - 2% of the seed culture is added to the culture medium) and the aerobic submerged cultivation is carried out.
The blow amount of sterile air supplied to the culture medium varies according to the type of the fermentation tank but is usually 0.2-1.0 V V M (i.e., 0.2-1.0 volume of air per volume of culture per minute) for obtaining the sufficient accumulation of DE-3936.
Substance DE-3936 can be collected from the fermentation broth as the form of free acid or a salt thereof by an ordinary manner. That is, since the free acid of DE-3936 or salts thereof is soluble in various organic solvents, the product is first extracted from the fermentation broth with a proper organic solvent and then is purified by chromatography, recrystallization, ~ etc. In this case, it is preferable that the extraction is carried out after adjusting the pH of the fermenation broth to 7.0-8.0 using, for example, an aqueous sodium hydroxide solution.
-~ However, in many cases, the pH of the broth after the fermentation is in the aforesaid range, so the control of pH may be not necessary. The extraction is carried out usually after separating - the fermentation broth into mycelial cake and filtrate broth by filtration both of which contain DE-3936. The antibiotic is - extracted, for example, with methanol or acetone from the cake and with benzene, chloroform, dichloroethane or ethyl acetate from the filt_ate broth. Then, each solvent layer containing DE-3936 is separated from the aqueous layer or the solid and these solvent layers are combined. After concentrating by _ g _ -~0644Z0 evaporation under a reduced pressure, the mixture is purified by, for example, a column chromatography containing alumina.
n this case, benzene, ethyl acetate, methanol, or a mixed solvent of them is used as the developing solvent.
The eluate is divided in to fractions and the fractions containing DE-3936 are monitored with antimicrobial activity or color reaction (vanilin-H2S04) and desirable fractions are collec~1 The fraction thus collected is concentrated under a reduced pressure and is further purified by the proper combination of a column chromatography of Cephadex* LH-20 (methanol is proper as the developed solvent), or silica gel (benzene, acetone or a mixture of them is suitable as the developing solvent), and the - treatment with activated carbon. The fractions containing DE-3936 are collected and concentrated under a reduced pressure to provide white powder of DE-3936. The powder may be crystallized by a conventional manner. DE-3936 is dissolved in a small amount of, for e~ample, acetone and after the addition of petroleum ether thereto, the mixture is allowed to stand at room temperature or ` in a cold place to provide the colorless prisms or pillars of DE-3936. The crystal obtained is a complex or composition of the salt of DE-3936, which consists of mainly sodium salt thereof and in addition potassium salt and ammonium salt. The ratio of the components in the product may vary according to the condition of the fermentation.
The antibiotic, DE-3936,thus obtained can be converted to any of the required form such as free acid, sodium salt, potassium salt, ammonium salt, silver salt, etc. by the following manner. The substance DE-3936 obtained from the fermentation broth is dissolved in an organic solvent immiscible with water, such as benzene, chloroform, etc., and the solution is shaken with diluted hydrochloric acid. This operation is repeated several * Trade Mark 1 times, the solvellt layer is then separated and concentrated to provide the white powder of the free acid of D~-3936. sy re-crystallizing the white powder thus obtained from benzene or a petroleum solvent, the pure free acid of DE-3936 is obtained as the colorless pillars or prisms.
Alternatively, the free acid can be obtained by another method. That is, the substance DE-3936 is dissolved in acetone and a proper amount of water is added to the solution, then the pH of the mixture is adjusted to 2-4 with diluted hydrochloric acid, and then the substance is extracted with a water-immiscible solvent such as benzene and the like.
DE-3936 may be also ob~ained as the sodium salt, potassium salt, or ammonium salt by the following manner. That is, the free acid of the substance DE-3936 is dissolved in acetone, to the solution a proper amount of water is added and the pH of the solution is adjusted to 9 - 12 by adding thereto an aqueous solu-tion of sodium hydroxide, potassium hydroxide, or ammonia, and after removing acetone from the mixture by concentration, ~he substance is extracted with a solvent such as benzene, ethyl acetate, chloroform, etc., followed by concentration, whereby ..
~; the corresponding salt of DE-3936 is obtained as the colorless powder. Furthermore, by recrystallizing the colorless powder thus obtained from a suitable solvent system such as benzene-petroleum ether, benzene-n-hexane, or water-methanol, the salt of DE-3936 is obtained as needles, pillars, or prisms.
Also, the silver salt of substance DE-3936 is obtained -~by dissolving the sodium salt or potassium salt of DE-3936 in methanol and then adding to the solution an aqueous solution of silver nitrate. Furthermore, by recrystallizing the silver salt of DE-3936 from a ben~ene-petroleum ether or a water-methanol, ~ ...
. -- 11 --~, '' : , ' ~
1 the silver salt of DE-3936 is obtained as the pillars or prisms.
The sodium salt of DE-3936 obtained by recrystallizing from benzene-pe-troleum ether possesses the following physical and chemical properties:
1. Appearance of the substance:
Colorless crystals in the for~ of needles, pillars or prisms, which are almost neutral as the sodium salt of the monocar-boxylic acid.
2. Melting point:
173.0 - 176.0C. (by a capillary method). Since the sodium salt melts with slightly turning to brown the salt is considered to melt with decomposition.
3. Optical rotation:
[~]25 ~ 67.0 (C = 1, chloroform).
4. Solubility: Easily soluble in lower alcohols, esters, , chloroform, carbon etrachloride, ethers, benzene, etc.;
soluble in cyclohexane, n-hexane, petroleum ether, etc.; but almost insoluble in water.
5. Elementary analysis:
C 62.52% H 8.90% Na 2.51%
~- 6. Color reaction:
Positive in sulfuric acid reaction, phosphoric acid reaction, p-anisaldehyde reaction, antimony trichloride reaction, iodine reaction, and vanillin-sulfuric acid reaction. Negative in Molisch's reaction, anthrone reaction, and ninhydrin re-action.
7. Ultraviolet absorption spectrum: The sodium salt does not show the specific absorption peak in the range of - 210 - 370 n.m. when measured in methanol.
8. Infrared absorption spectrum (in chloroform):
As shown in Fig. 1.
1 9. Nuclear magnetic resonance spectrum (in CDC13):
- Spectrum (Fig. 2) shows that the substance possesses four methoxy groups. Molecular weight and molecular formula of DE-3936 have been clari~ied by mass spectrometry of methyl ~ -ester of DE-3936 and high resolution mass spectrometry of ,` the free acid thereof to be C~4H75C14Na and 850 (851.07 when calculated from the International Atomic Weight), respectively.
From these data and the result of X-ray diffractiometry, the chemical structure of DE-3936 was determined as shown in Fig. 3.
Furthermore, the biological properties of the sodium salt of DE-3936 are as follows: `
1. Antimicrobial spectrum:
The sodium salt of DE-3936 possesses an antimicro~ial activity to gram positive bacteria but does not show anti-microbial activity to gram negative bacteria, molds, and yeast. Antimicrobial spectrum is shown in Table 3.
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~ 20 ,~ . ..
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:
:
.- ....... , : , .
.: ~ ..... .
1 TABLE 3 Antimicrobial spectrum of antibiotic DE-3936 sodium salt .
Test OrganismMIC (mcg/ml) Medium Staphylococcus aureus 209 p 12.5 Sta~hylococcus aureus Smith 6.25 :
Staphylococcus aureus 393.12 (PC-G,SM,TC,Macrolides-R) Streptococcus faecalis ATCC-8043 1.56 Streptococcus pneumoniae DP-l 1.56 ~acillus subtilis ATCC-6633 3.12 - ------Sarcina lutea ATCC-93413.12 Mycobacterium ATCC 60712.5 Escherichia coli NIHJ< 100 Pseudomonas aeruginosa No. 15 < 100 _ _ Mycoplasma hominis DH 100 Mycoplasma gallisepticum GP-31 25 Mycoplasma pulmonis M-53 50 Piricularia oryzae < 100 II
Tricho~hyton mentagrophytes < 100 II
Candida aIbicans < 100 II
.~ ~
* MIC was determined by agar dilution method.
~; ** Medium I; Heart infusion agar. Medium II; Sabouraud's agar ' 2. Acute toxicity:
~1) LD50 to mouse (days strain, male) is as follows:
45.8 mg/kg (oral administration) , 13.0 mg/kg (interperitoneal administration).
(2) LD50 to chicken (2-3 weeks of age, male) is as follows:
^:
190.0 mg/kg (oral administration) 1.13 mg/kg (interperitoneal administration).
. ~
.... . .
; - 14 -. , .
1 The chemical properties of the free acid of DE-3936 are as follows:
1. Appearance of the substance:
Colorless prisms which shows acidic and turns to yellow by BPs.
2. Melting point:
109 - 114C (capillary method).
- 3. Optical rotation:
[]D ~ 66.6C (C = 1, chloroform3.
4 Elementary analysis:
`~ C 64.65% H 9.05%
The solubility, color reaction, and ultraviolet absorp-tion spectra of the acid are almost same as those of the sodium ; salt.
The new antibiotic, DE-3936, is found to be effective on proto~oal diseases in animals such as malaria, toxoplasmosis, ;~
coccidiosis and the like. That is, the antibiotic can prevent from or cure the diseases, moreover, the drug may be expected to produce synergic effect with other drugs such as sulfa drugs, folic acid antagonists, quinoline derivatives, anti-thiamines which have now been used to the protozoal diseases in animals.
For example, when DE-3936 or its salt was administered ; to the mice of over 15 mg/kg body weight (one or several times) which had been infected with Plasmodium berghei NH-65 or Toxo-plasma gondii RH, the drug apparently prolonged the life of mice ~; as compared with the controls which were not medicated. If the antibiotic is administered to animals one or two days prior to the infection, the effect would be more remarkable. Thus the antibiotic, DE-3936, is useful for curing or preventing from malaria and toxoplasmosis in animals.
.~ .
:` . .
~- :
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.. - , , , , ~
Moreover, DE 3936 or its salt is very effective on coccidiosis in animals. For example, the animal feed containing 25 - 150 ppm of DE-3936 or salt thereof p~events animals from the infection of Eimeria tenella, Eimeria necatrix, Eimeria acervulina, Eimeria maxima or mixed infection of them. When the antibiotic was mixed with feed at a concentration of 100 -500 ppm, remarkable curing effect is recognized in these proto-zoal diseases. Particularly, this new antibiotic is ef~ective to the diseases caused by resistant strains to sulfa drugs, 1 quinoline derivatives or antithiamines.
The effect of DE-3936 on coccidiosis in chickens was confirmed by the following experiments.
Chickens of 8 days old were divided into three groups (ten chickens a group), i.e. the normal control group, the infected control group and the medicated group, and bred with the feed having the composition;
; corn 47% lucerne meal 3%
barley flour 10% calcium carbonate 3%
defatted rice bran 10% sugar 2%
soybean meal 10~ sodium chloride 0.5%
fish meal 7% vitamin F 0.5%
wheat bran 7%
The chickens of normal control were not infected with coccidiosis and not medicated with the drug. The infected control includes chickens which were infected but not medicated and the medicated group means the group of chickens which were infected and medicated. The medication was made by breeding with the feed containing DE-3936 or its salt and the infection with coccidiosis was made by inoculating sporated oocysts of Eimeria protozoa after 48 hours from the start of breeding. Since the test period varies depending upon the kind of coccidial protozoa, ' .
` ~0649~Z0 1 the evaluation was made at the fourth to seventh day a~ter infection. The results are shown in the Tables 4 to 8. , In the tables, weight gain and feed efficiency are represented by the relative values to that of normal control and feed efficiency means weight gain per consumed feed weight.
OPG means oocyst per gram of feces.
Effect of DE-3936 to the infection be Eimeria tenella (5 x 104) (Results after 7 days from the infections) Concentration Morta- Feed effi- Weight of DE-3936 in lity ciency gainO.P.G.
the feed (ppm) (%) (~) (~
.. .... .. .. _ Normal , control _ 0 100 100 0 . .. . .. , ~ ..
Medicated 25 10 61 68 2.0x104 0 81 903.5x103 .
~.:
,., . . _ . -Infected control _ 50 -31 -23 2.6x106 . _. ___ .._ l . ., -`~ TABLE 5 _____ ~
Effect o~ DE-3936 to the infection by Eimeria necatrix ~ (5 x 104) (Results after 7 days from the infection) ; Concentration Morta- Feed effi- Weight of DE-3936 in lity ciency gainO.P.G.
the feed (ppm) (%) (~) (%) .' --- . .. _ _ _ .. . __ ._ Normal control _ 0 100 100 0 ~ ... .. . . _ _. __ . _ ~edicated25 20 26 43 2 9x105 0 80 88 4 4x103 __~ ., _ , Infected ~- control _ 100 _ _ ' ~ .. _ ... _ _. . .__ ~, .
`` ~064420 Effect of DE-3936 to the infection by Eimeria acervulina (1 x 105) (Results after 4 days from the infection) Concentration Morta- Feed effi- Weight of DE-3936 in lity ciency gain O.P.G.
the feed (ppm) (%) (~) ( Q ) .__ ._ ..
Normal control _ 0 100 100 0 . .
Medicated 25 0 90 93 7.9x105 0 106 107 4.8x104 0 108 106 1.6x103 -- ..... _ .. . _ ._ Infected 7 control _ . . 0 92 102 1.2xlO
- ___. _ , ' , .
Effect of DE-3936 to the infection by Eimeria maxima (6.2 x 104) (Results after 5 days from the infection~
I Concentration Morta- Feed effi- Weight i~ of DE-3936 in lity ciency gain O.P.G.
the feed (ppm~ (%~ (~) (~) .
.. Normal ;; control - . 0 100 . 100 0 . _ Medicated 25 0 93 93 5.2x103 0 `101 107 1.3x103 0 98 102 0 :
'~ 100 0 102 104 0 . 125 . 0 99 102 0 ::
:, . . . ._._ .
; Infected 5 -."' control _ . _ _ 87 6.3xl0 :`"" ' ': :
, ~
,."' ,.
, - :
' `-" 10644Z0 .
Ef~ect of DE-3936 to mixed infection by E. tenella, (5 x 104~, E. necatrix (5 x 10 ), and E. acervulina (1 x 106) (Results after 7 days from the infection) Concentra~on Morta- Feed effi- Weight of DE-3936 in lity ciency gain ~-the feed ~ppm~ (%) ~%) (%) ._ . ._ ~
Normal control _ 0 100 100 .. _ .... _ Medicated 25 40 -4 23 ~- 50 10 S9 67 . . .....
Infected control _ 100 _ ' _ Out of the known polyether antibiotics, monensin has now been used in the United States as anticoccidial agent but from the results shown in "Advanced Pharmacology ~ Chemotherapy", ' page 241 (1973~ and the comparison tests made by the present inventors, the anticoccidial activity of DE-3936 is superior to that of monensin.
- 20 That is, according to the results in Table IV on page 241 of the aboYe-mentioned literature, death of chicken was re-- ported even in 250 ppm administered group, though the chickens were infected with 2 x 105 oocystes. In the 250 ppm group, oocysts still remained in feces at 4.37 x 10 /chick and average weight gain was 36.9 g, whereas that of the normal control showed 59 g.
.. . .
On the other hand,the present in~entor investigated the effect of DE-3936 compared with monensin by breeding chickens of 8 days old with the feed containing the drug, after the infection with multi-resistant oocysts of E. necatrix (3.4 x 104).
The results are shown in Table 9.
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, . . .
'.: . ~ ' ': . , ` ~0644Z0 Comparison test results of DE-3936 and monensin to multi~resistant strain of E. necatrix Concen- Weight Feeding Abnor- Disorders tration Morta- gain effi- mality in the small (ppm) lity (%) ciency in intestines _ _ _ (%)feces Normal control 0/5100 100 DE-3936 75 0/5105 103 _ medicated 1000/5 104 102 125 0/5101 100 _ -- --Monensin75 2/5 21 58 ++++ ++++
medicated 1001/5 51 77 +++ ++++
125 0/5 86 89 ++ +++
. ._ Infected control _ 4/5 -17 37 ++++ ++++
.,-- . ._ _ ... ,~ . .
The indexes mean the degree of abnormality or disorders i.e. - means normal and +, ++, +++, and ++++ means from slight degree to heavy in turn. -As seen in the table, two cf five chickens and one of five chicke~ died in 75 ppm and 100 ppm monensin group, respect-ively. Since three of five chickens died in the non-medicated group, monensin may have some effect but the effect is quite ;
.. ..
low as compared with DE-3936.
Moreover, disorders in the intestinal region were observed in monensin groups but not in DE-3936 groups and ab-normality in the feces (bloody excrement, mix of mucous membrane or disordered digestion) were remarkable found in the monensin grOup and the non-medicated control, however, not found in the DE-3936 group.
As described in detail as above, the new antibiotic, DE-3936, is a very useful antiprotozoal drug for animals, especially it is effective against coccidiosis in chicken.
:
: ' . .
' .~-.' ' ' '', , ~,', ' , .
1 In the above experimentation, DE-3936 was used as its complex form consisting mainly of sodi~m salt, however, data obtained by the use of pure sodium salt, potassium salt, ammonium salt and free acid of DE-3936 were almost the same as the above shown data. Therefore, any salt or free acid of DE-3936, or complex thereof can be used for the prevention or treatment of coccidiosis in animals. For the administration to the animal, it is preferable to mix the drug with the feed, however, any other preparation used in veterinary field such as aqueous solution, powder, capsules, tablets, emulsion, or injection may be used according to the purpose or condition.
The following examples illustrate the procedure of fermentation and isolation of DE-3936.
EXAMPLE l Production of substance DE-3936 by means of rotary shaking incubator:
A seed culture was prepared by cultivating Streptomyces ; hygroscopicus No. 9735-1 in a pre-culture medium having the following composition at 28C for 3 days.
glucose 20 g/liter meat extract 3 g/liter peptone 5 g/liter ~; dry yeast 3 g/liter sodium chloride 5 g/liter calcium carbonate 3 g/liter pH 7.0 - The main cultivation was carried out using the culture medium of the following composition.
glucose 30 g/liter cotton seed flour 5.5 g/liter (pharmamedia) ' .
" 1064420 1 sodium chloride 5 g/liter calcium carbonate 3 g/liter pH 7.0 In a 300 ml Erlenmeyer flask was placed 40 ml of the above-described culture medium and after inoculating 0.8 ml of the see~ culture prepared above, the cultivation was carried out 28C and at 200 r.p.m. for 6 days. After the cultivation, the - culture medium was filtered. When the antimicrobial activity of the filtrate was measured by bioassay, it WaS found that substance DE-3936 was produced in an amount of 510 mcg/ml.
Moreover, it was found that the substance existed in the mycelium as well. Then, 50 g of wet mycelial cake obtained from one liter of the broth was extracted twice with 200 ml methanol, the extract was concentrated to about 50 ml. After the addition of 200 ml of water, the mixture was extracted twice with 100 ml of benzene.
On the other hand, the filtrate from the broth was extracted twice with 250 ml of benzene and the extract was combined with ~ ,' the benzene extract from the mycelial cake. The mixed extract was concentrated and an oily substance with reddish brown was obtained. The substance was dissolved in methanol and after removing insoluble materials by filtration, the filtrate was subjected to a column chromatography of Cephadex*LH-20 followed by development with methanol. After repeating twice the operation, the fractions containing antibiotic DE-3936 were collected and concentrated to dryness to give 750 mg of a brown powder. Then, for discoloring, the powder was dissolved in 30 ml of benzene, the solution was passed through a column packed with activated carbon ta mixture of 1 g of activated carbon and 1 g of sellaite), then the column was washed sufficiently with benzene. The eluate - 30 was concentrated to dryness and 680 mg of the white powder of DE-3936.
* Trade Mark , - -: , . : ~
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.
`"` 10644~0 1 EXA~LE 2 Production of substance DE-3936 b~ means of 200-liter fermentation tank:
A producing culture medium having the following compo-sition was used.
glucose 25 g/liter cotton seed flour 5 g/liter corn steep liquor 5 g/liter sodium chloride 5 g/liter ; 10 calcium carbonate 3 g/liter antifoaming agent 30 ml/130 liters pH 6.8 In 200-liter fermentation tank was placed 130 liters of the medium and one liter of the seed culture prepared by the same manner as in Example 1 was inoculated. The fermentation was carried out for 96 hours at 30C, with aeration of 70 liters/
minutes, under an inside pressure of 1 kg/cm2, and with stirring ` at 200 r.p.m.... Then the fermentation broth was filtered and about 2.6 kg of wet mycelial ca~e and about 120 liters of filtrate (pH 7.0 and showing DE~3936 potency of 310 mcg/ml) were obtained.
The wet mycelial cake was extracted once with 15 liters and then additionally with 5 liters of methanol. The methanol extract was concentrated to about 2 liters to remove methanol and 1 liter of water was added to the residue followed by extraction four times with 3 liters of benzene. On the other hand, the filtrate was extracted once with 35 liters of benzene and then with additional 20 liters of benzene. The benzene extracts thus -recovered from the mycelial cake and the filtrate were combined and concentrated, so about 200 g of a dark red oily material was obtained. The oily material was first purified by means of active `` 1064420 1 alumina column chromatography. That is, the benzene solu-tion of the oily material was passed through the column, and impurities were removed by eluting with benzene as completely as possible.
Then the column was washed with ethyl acetate and then a mixture of ethyl acetate and methanol ~l : 1). The fractions containing active material were concentrated and dried to provide a reddish brown oily material. Then the material was ;
dissolved in methanol and the solution was passed through a column of Cephadex* LH-20. The adsorbed material was developed 10 with methanol, and the active fractions obtained were con- ~-centrated to provide 55 g of a reddish brown oily material.
The material was further purified by a silica gel column chro-matography. That is, ~5 g of the reddish brown oily material thus recovered was first dissolved in benzene, the solution was passed through the column, and after removing impurities by eluting the column with benzene and then a mixture of acetone and benzene ~1:50), the active material was eluted with a mixture of acetone and benzene ~1 : 20 to 1 : 5). After repeating twice the operation, the eluate was concentrated to dryness to provide 31.5 g of DE-3936 as the pale yellowish powder.
. _.
Crystallization of DE-3936:
The crystallization was carried out using 6.8 g of the powder of DE-3936 obtained by the processes shown in Examples l and 2. The powder was dissolved in lO ml of benzene, whereby the prism~like crystals of DE~3936 began to form immediately and after the addition of 10 ml of petroleum ether, the mixture was allowed to stand for 3 hours in a refrigerator at 5C. Then the crystals thus precipitated were separated from the mother *Trade MarR
` 10644Z0 1 liquor and the crystals were washed a few times with petroleum ether. The wash ether was added to the mother liquor and the mixture was allowed to stand in a refrigerator to precipitate secondary crystals. The crystals were separated and washed with petroleum ether. The wash ether was combined with the mother liquor and crystallization was carried out again from the mixture to provide third crystals. The yields for the crystals thus recovered were 4.6 g for the first crop, 0.3 g for the second crop, and 0.2 g for third crop. That is, 5.1 g in total of the colourless prism-like crystals of DE-3936 was obtained.
Crystallization and recrystallization of DE-3936:
In 10 ml of benzene was dissolved 2.8 g of the powder of DE-3936 obtained by the processes shown in Examples 1 and 2 and the solution was allowed to stand at room temperature, whereby colourless prism-like crystals were precipitated. The crystals were collected by filtration, washed a few times with petroleum ether, and the washed ether was added to the mother liquor. The mixture was concentrated to dryness and the residue was dissolved in 5 ml of benzene. The solution was allowed to - stand at room temperature to precipitate crystals, which were collected by filtration, and 1 g of the colourless prism of DE-3936 was obtained. The mother liquor was concentrated to dryness and the residue was dissolved in 10 ml of benzene and 30 ml of petroleum ether was added to the solution, whereby 1 g of crystals in the form of colourless prism were obtained. Then, 1 g of the crystals thus obtained were dissolved in 6 ml of methanol and after adding 1.5 ml of water to the solution, the mixture was allowed to stand at room temperature to -precipitate colourless needles, which were collected by filtration 1 to provide 360 mg of the crystals. Also, by allowing to stand the mother liquor at room temperature, 160 mg oE the prisms were obtained.
In addition, in the case of forming crystals of ;~
- DE-3936, needles or fine prisms were obtained when the crystalli-zation was carried out in a short period of time, while the large prisms were obtained when the crystallization was carried out gradually.
1 9 ,:, -Preparation of the free acid of DE-3936: ;
In 100 ml of acetone was added 1 g of DE-3936 obtained in Example 3 and then 50 ml of water was added to the solution, pH of which was about 7.2. After adjusting the pH of the solu-tion to 2.9 with 0.5 normal hydrochloric acid, the product was extracted three times with 500 ml of benzene. The extract was washed with water, dehydrated with sodium sulfate, and concentrated to dryness to give the 1 g of white powder. The powder was dissolved in 8 ml of benzene, and the solution was concentrated to about 2 milliliters, and after adding 6 ml of petroleum ; ether thereto, the mixture was allowed to stand at room tem-perature for 2 days, whereby colorless prisms were formed.
The crystals were recovered by filtration and washed with petroleum ether to provide 800 mg of the colorless prisms of DE-3936.
Preparation of the sodium salt of DE~3936~
In 50 ml of acetone was dissolved 1 g of the powder - of free E-3936 obtained in Example S and then 25 ml of water was added to the solution. After adjusting the pH of the solution to 12.2 by adding 0.5 normal aqueous potassium hydroxide solution, .. . , . . . : . : ..
`` ~064420 acetone was distilled off. The residual aqueous solution was extracted with ethyl acetate and the extract was washed with water, dehydrated with sodium sulfate,and concentrated to dryness to provide about 90 mg of white powder. In 10 ml of benzene - was dissolved S00 mg of the powder thus obtained and the solution was concentrated to about 1 milliliter. Then, 5 ml of petroleum ether was added to the concentrate and the mixture was allowed to stand at room temperature, whereby colourless prisms were precipitated. By collecting the crystals, 390 mg of the potassium salt of DE-3936 was obtained.
:",' ` ' ': :
C 62.52% H 8.90% Na 2.51%
~- 6. Color reaction:
Positive in sulfuric acid reaction, phosphoric acid reaction, p-anisaldehyde reaction, antimony trichloride reaction, iodine reaction, and vanillin-sulfuric acid reaction. Negative in Molisch's reaction, anthrone reaction, and ninhydrin re-action.
7. Ultraviolet absorption spectrum: The sodium salt does not show the specific absorption peak in the range of - 210 - 370 n.m. when measured in methanol.
8. Infrared absorption spectrum (in chloroform):
As shown in Fig. 1.
1 9. Nuclear magnetic resonance spectrum (in CDC13):
- Spectrum (Fig. 2) shows that the substance possesses four methoxy groups. Molecular weight and molecular formula of DE-3936 have been clari~ied by mass spectrometry of methyl ~ -ester of DE-3936 and high resolution mass spectrometry of ,` the free acid thereof to be C~4H75C14Na and 850 (851.07 when calculated from the International Atomic Weight), respectively.
From these data and the result of X-ray diffractiometry, the chemical structure of DE-3936 was determined as shown in Fig. 3.
Furthermore, the biological properties of the sodium salt of DE-3936 are as follows: `
1. Antimicrobial spectrum:
The sodium salt of DE-3936 possesses an antimicro~ial activity to gram positive bacteria but does not show anti-microbial activity to gram negative bacteria, molds, and yeast. Antimicrobial spectrum is shown in Table 3.
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1 TABLE 3 Antimicrobial spectrum of antibiotic DE-3936 sodium salt .
Test OrganismMIC (mcg/ml) Medium Staphylococcus aureus 209 p 12.5 Sta~hylococcus aureus Smith 6.25 :
Staphylococcus aureus 393.12 (PC-G,SM,TC,Macrolides-R) Streptococcus faecalis ATCC-8043 1.56 Streptococcus pneumoniae DP-l 1.56 ~acillus subtilis ATCC-6633 3.12 - ------Sarcina lutea ATCC-93413.12 Mycobacterium ATCC 60712.5 Escherichia coli NIHJ< 100 Pseudomonas aeruginosa No. 15 < 100 _ _ Mycoplasma hominis DH 100 Mycoplasma gallisepticum GP-31 25 Mycoplasma pulmonis M-53 50 Piricularia oryzae < 100 II
Tricho~hyton mentagrophytes < 100 II
Candida aIbicans < 100 II
.~ ~
* MIC was determined by agar dilution method.
~; ** Medium I; Heart infusion agar. Medium II; Sabouraud's agar ' 2. Acute toxicity:
~1) LD50 to mouse (days strain, male) is as follows:
45.8 mg/kg (oral administration) , 13.0 mg/kg (interperitoneal administration).
(2) LD50 to chicken (2-3 weeks of age, male) is as follows:
^:
190.0 mg/kg (oral administration) 1.13 mg/kg (interperitoneal administration).
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; - 14 -. , .
1 The chemical properties of the free acid of DE-3936 are as follows:
1. Appearance of the substance:
Colorless prisms which shows acidic and turns to yellow by BPs.
2. Melting point:
109 - 114C (capillary method).
- 3. Optical rotation:
[]D ~ 66.6C (C = 1, chloroform3.
4 Elementary analysis:
`~ C 64.65% H 9.05%
The solubility, color reaction, and ultraviolet absorp-tion spectra of the acid are almost same as those of the sodium ; salt.
The new antibiotic, DE-3936, is found to be effective on proto~oal diseases in animals such as malaria, toxoplasmosis, ;~
coccidiosis and the like. That is, the antibiotic can prevent from or cure the diseases, moreover, the drug may be expected to produce synergic effect with other drugs such as sulfa drugs, folic acid antagonists, quinoline derivatives, anti-thiamines which have now been used to the protozoal diseases in animals.
For example, when DE-3936 or its salt was administered ; to the mice of over 15 mg/kg body weight (one or several times) which had been infected with Plasmodium berghei NH-65 or Toxo-plasma gondii RH, the drug apparently prolonged the life of mice ~; as compared with the controls which were not medicated. If the antibiotic is administered to animals one or two days prior to the infection, the effect would be more remarkable. Thus the antibiotic, DE-3936, is useful for curing or preventing from malaria and toxoplasmosis in animals.
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Moreover, DE 3936 or its salt is very effective on coccidiosis in animals. For example, the animal feed containing 25 - 150 ppm of DE-3936 or salt thereof p~events animals from the infection of Eimeria tenella, Eimeria necatrix, Eimeria acervulina, Eimeria maxima or mixed infection of them. When the antibiotic was mixed with feed at a concentration of 100 -500 ppm, remarkable curing effect is recognized in these proto-zoal diseases. Particularly, this new antibiotic is ef~ective to the diseases caused by resistant strains to sulfa drugs, 1 quinoline derivatives or antithiamines.
The effect of DE-3936 on coccidiosis in chickens was confirmed by the following experiments.
Chickens of 8 days old were divided into three groups (ten chickens a group), i.e. the normal control group, the infected control group and the medicated group, and bred with the feed having the composition;
; corn 47% lucerne meal 3%
barley flour 10% calcium carbonate 3%
defatted rice bran 10% sugar 2%
soybean meal 10~ sodium chloride 0.5%
fish meal 7% vitamin F 0.5%
wheat bran 7%
The chickens of normal control were not infected with coccidiosis and not medicated with the drug. The infected control includes chickens which were infected but not medicated and the medicated group means the group of chickens which were infected and medicated. The medication was made by breeding with the feed containing DE-3936 or its salt and the infection with coccidiosis was made by inoculating sporated oocysts of Eimeria protozoa after 48 hours from the start of breeding. Since the test period varies depending upon the kind of coccidial protozoa, ' .
` ~0649~Z0 1 the evaluation was made at the fourth to seventh day a~ter infection. The results are shown in the Tables 4 to 8. , In the tables, weight gain and feed efficiency are represented by the relative values to that of normal control and feed efficiency means weight gain per consumed feed weight.
OPG means oocyst per gram of feces.
Effect of DE-3936 to the infection be Eimeria tenella (5 x 104) (Results after 7 days from the infections) Concentration Morta- Feed effi- Weight of DE-3936 in lity ciency gainO.P.G.
the feed (ppm) (%) (~) (~
.. .... .. .. _ Normal , control _ 0 100 100 0 . .. . .. , ~ ..
Medicated 25 10 61 68 2.0x104 0 81 903.5x103 .
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,., . . _ . -Infected control _ 50 -31 -23 2.6x106 . _. ___ .._ l . ., -`~ TABLE 5 _____ ~
Effect o~ DE-3936 to the infection by Eimeria necatrix ~ (5 x 104) (Results after 7 days from the infection) ; Concentration Morta- Feed effi- Weight of DE-3936 in lity ciency gainO.P.G.
the feed (ppm) (%) (~) (%) .' --- . .. _ _ _ .. . __ ._ Normal control _ 0 100 100 0 ~ ... .. . . _ _. __ . _ ~edicated25 20 26 43 2 9x105 0 80 88 4 4x103 __~ ., _ , Infected ~- control _ 100 _ _ ' ~ .. _ ... _ _. . .__ ~, .
`` ~064420 Effect of DE-3936 to the infection by Eimeria acervulina (1 x 105) (Results after 4 days from the infection) Concentration Morta- Feed effi- Weight of DE-3936 in lity ciency gain O.P.G.
the feed (ppm) (%) (~) ( Q ) .__ ._ ..
Normal control _ 0 100 100 0 . .
Medicated 25 0 90 93 7.9x105 0 106 107 4.8x104 0 108 106 1.6x103 -- ..... _ .. . _ ._ Infected 7 control _ . . 0 92 102 1.2xlO
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Effect of DE-3936 to the infection by Eimeria maxima (6.2 x 104) (Results after 5 days from the infection~
I Concentration Morta- Feed effi- Weight i~ of DE-3936 in lity ciency gain O.P.G.
the feed (ppm~ (%~ (~) (~) .
.. Normal ;; control - . 0 100 . 100 0 . _ Medicated 25 0 93 93 5.2x103 0 `101 107 1.3x103 0 98 102 0 :
'~ 100 0 102 104 0 . 125 . 0 99 102 0 ::
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; Infected 5 -."' control _ . _ _ 87 6.3xl0 :`"" ' ': :
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Ef~ect of DE-3936 to mixed infection by E. tenella, (5 x 104~, E. necatrix (5 x 10 ), and E. acervulina (1 x 106) (Results after 7 days from the infection) Concentra~on Morta- Feed effi- Weight of DE-3936 in lity ciency gain ~-the feed ~ppm~ (%) ~%) (%) ._ . ._ ~
Normal control _ 0 100 100 .. _ .... _ Medicated 25 40 -4 23 ~- 50 10 S9 67 . . .....
Infected control _ 100 _ ' _ Out of the known polyether antibiotics, monensin has now been used in the United States as anticoccidial agent but from the results shown in "Advanced Pharmacology ~ Chemotherapy", ' page 241 (1973~ and the comparison tests made by the present inventors, the anticoccidial activity of DE-3936 is superior to that of monensin.
- 20 That is, according to the results in Table IV on page 241 of the aboYe-mentioned literature, death of chicken was re-- ported even in 250 ppm administered group, though the chickens were infected with 2 x 105 oocystes. In the 250 ppm group, oocysts still remained in feces at 4.37 x 10 /chick and average weight gain was 36.9 g, whereas that of the normal control showed 59 g.
.. . .
On the other hand,the present in~entor investigated the effect of DE-3936 compared with monensin by breeding chickens of 8 days old with the feed containing the drug, after the infection with multi-resistant oocysts of E. necatrix (3.4 x 104).
The results are shown in Table 9.
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'.: . ~ ' ': . , ` ~0644Z0 Comparison test results of DE-3936 and monensin to multi~resistant strain of E. necatrix Concen- Weight Feeding Abnor- Disorders tration Morta- gain effi- mality in the small (ppm) lity (%) ciency in intestines _ _ _ (%)feces Normal control 0/5100 100 DE-3936 75 0/5105 103 _ medicated 1000/5 104 102 125 0/5101 100 _ -- --Monensin75 2/5 21 58 ++++ ++++
medicated 1001/5 51 77 +++ ++++
125 0/5 86 89 ++ +++
. ._ Infected control _ 4/5 -17 37 ++++ ++++
.,-- . ._ _ ... ,~ . .
The indexes mean the degree of abnormality or disorders i.e. - means normal and +, ++, +++, and ++++ means from slight degree to heavy in turn. -As seen in the table, two cf five chickens and one of five chicke~ died in 75 ppm and 100 ppm monensin group, respect-ively. Since three of five chickens died in the non-medicated group, monensin may have some effect but the effect is quite ;
.. ..
low as compared with DE-3936.
Moreover, disorders in the intestinal region were observed in monensin groups but not in DE-3936 groups and ab-normality in the feces (bloody excrement, mix of mucous membrane or disordered digestion) were remarkable found in the monensin grOup and the non-medicated control, however, not found in the DE-3936 group.
As described in detail as above, the new antibiotic, DE-3936, is a very useful antiprotozoal drug for animals, especially it is effective against coccidiosis in chicken.
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1 In the above experimentation, DE-3936 was used as its complex form consisting mainly of sodi~m salt, however, data obtained by the use of pure sodium salt, potassium salt, ammonium salt and free acid of DE-3936 were almost the same as the above shown data. Therefore, any salt or free acid of DE-3936, or complex thereof can be used for the prevention or treatment of coccidiosis in animals. For the administration to the animal, it is preferable to mix the drug with the feed, however, any other preparation used in veterinary field such as aqueous solution, powder, capsules, tablets, emulsion, or injection may be used according to the purpose or condition.
The following examples illustrate the procedure of fermentation and isolation of DE-3936.
EXAMPLE l Production of substance DE-3936 by means of rotary shaking incubator:
A seed culture was prepared by cultivating Streptomyces ; hygroscopicus No. 9735-1 in a pre-culture medium having the following composition at 28C for 3 days.
glucose 20 g/liter meat extract 3 g/liter peptone 5 g/liter ~; dry yeast 3 g/liter sodium chloride 5 g/liter calcium carbonate 3 g/liter pH 7.0 - The main cultivation was carried out using the culture medium of the following composition.
glucose 30 g/liter cotton seed flour 5.5 g/liter (pharmamedia) ' .
" 1064420 1 sodium chloride 5 g/liter calcium carbonate 3 g/liter pH 7.0 In a 300 ml Erlenmeyer flask was placed 40 ml of the above-described culture medium and after inoculating 0.8 ml of the see~ culture prepared above, the cultivation was carried out 28C and at 200 r.p.m. for 6 days. After the cultivation, the - culture medium was filtered. When the antimicrobial activity of the filtrate was measured by bioassay, it WaS found that substance DE-3936 was produced in an amount of 510 mcg/ml.
Moreover, it was found that the substance existed in the mycelium as well. Then, 50 g of wet mycelial cake obtained from one liter of the broth was extracted twice with 200 ml methanol, the extract was concentrated to about 50 ml. After the addition of 200 ml of water, the mixture was extracted twice with 100 ml of benzene.
On the other hand, the filtrate from the broth was extracted twice with 250 ml of benzene and the extract was combined with ~ ,' the benzene extract from the mycelial cake. The mixed extract was concentrated and an oily substance with reddish brown was obtained. The substance was dissolved in methanol and after removing insoluble materials by filtration, the filtrate was subjected to a column chromatography of Cephadex*LH-20 followed by development with methanol. After repeating twice the operation, the fractions containing antibiotic DE-3936 were collected and concentrated to dryness to give 750 mg of a brown powder. Then, for discoloring, the powder was dissolved in 30 ml of benzene, the solution was passed through a column packed with activated carbon ta mixture of 1 g of activated carbon and 1 g of sellaite), then the column was washed sufficiently with benzene. The eluate - 30 was concentrated to dryness and 680 mg of the white powder of DE-3936.
* Trade Mark , - -: , . : ~
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`"` 10644~0 1 EXA~LE 2 Production of substance DE-3936 b~ means of 200-liter fermentation tank:
A producing culture medium having the following compo-sition was used.
glucose 25 g/liter cotton seed flour 5 g/liter corn steep liquor 5 g/liter sodium chloride 5 g/liter ; 10 calcium carbonate 3 g/liter antifoaming agent 30 ml/130 liters pH 6.8 In 200-liter fermentation tank was placed 130 liters of the medium and one liter of the seed culture prepared by the same manner as in Example 1 was inoculated. The fermentation was carried out for 96 hours at 30C, with aeration of 70 liters/
minutes, under an inside pressure of 1 kg/cm2, and with stirring ` at 200 r.p.m.... Then the fermentation broth was filtered and about 2.6 kg of wet mycelial ca~e and about 120 liters of filtrate (pH 7.0 and showing DE~3936 potency of 310 mcg/ml) were obtained.
The wet mycelial cake was extracted once with 15 liters and then additionally with 5 liters of methanol. The methanol extract was concentrated to about 2 liters to remove methanol and 1 liter of water was added to the residue followed by extraction four times with 3 liters of benzene. On the other hand, the filtrate was extracted once with 35 liters of benzene and then with additional 20 liters of benzene. The benzene extracts thus -recovered from the mycelial cake and the filtrate were combined and concentrated, so about 200 g of a dark red oily material was obtained. The oily material was first purified by means of active `` 1064420 1 alumina column chromatography. That is, the benzene solu-tion of the oily material was passed through the column, and impurities were removed by eluting with benzene as completely as possible.
Then the column was washed with ethyl acetate and then a mixture of ethyl acetate and methanol ~l : 1). The fractions containing active material were concentrated and dried to provide a reddish brown oily material. Then the material was ;
dissolved in methanol and the solution was passed through a column of Cephadex* LH-20. The adsorbed material was developed 10 with methanol, and the active fractions obtained were con- ~-centrated to provide 55 g of a reddish brown oily material.
The material was further purified by a silica gel column chro-matography. That is, ~5 g of the reddish brown oily material thus recovered was first dissolved in benzene, the solution was passed through the column, and after removing impurities by eluting the column with benzene and then a mixture of acetone and benzene ~1:50), the active material was eluted with a mixture of acetone and benzene ~1 : 20 to 1 : 5). After repeating twice the operation, the eluate was concentrated to dryness to provide 31.5 g of DE-3936 as the pale yellowish powder.
. _.
Crystallization of DE-3936:
The crystallization was carried out using 6.8 g of the powder of DE-3936 obtained by the processes shown in Examples l and 2. The powder was dissolved in lO ml of benzene, whereby the prism~like crystals of DE~3936 began to form immediately and after the addition of 10 ml of petroleum ether, the mixture was allowed to stand for 3 hours in a refrigerator at 5C. Then the crystals thus precipitated were separated from the mother *Trade MarR
` 10644Z0 1 liquor and the crystals were washed a few times with petroleum ether. The wash ether was added to the mother liquor and the mixture was allowed to stand in a refrigerator to precipitate secondary crystals. The crystals were separated and washed with petroleum ether. The wash ether was combined with the mother liquor and crystallization was carried out again from the mixture to provide third crystals. The yields for the crystals thus recovered were 4.6 g for the first crop, 0.3 g for the second crop, and 0.2 g for third crop. That is, 5.1 g in total of the colourless prism-like crystals of DE-3936 was obtained.
Crystallization and recrystallization of DE-3936:
In 10 ml of benzene was dissolved 2.8 g of the powder of DE-3936 obtained by the processes shown in Examples 1 and 2 and the solution was allowed to stand at room temperature, whereby colourless prism-like crystals were precipitated. The crystals were collected by filtration, washed a few times with petroleum ether, and the washed ether was added to the mother liquor. The mixture was concentrated to dryness and the residue was dissolved in 5 ml of benzene. The solution was allowed to - stand at room temperature to precipitate crystals, which were collected by filtration, and 1 g of the colourless prism of DE-3936 was obtained. The mother liquor was concentrated to dryness and the residue was dissolved in 10 ml of benzene and 30 ml of petroleum ether was added to the solution, whereby 1 g of crystals in the form of colourless prism were obtained. Then, 1 g of the crystals thus obtained were dissolved in 6 ml of methanol and after adding 1.5 ml of water to the solution, the mixture was allowed to stand at room temperature to -precipitate colourless needles, which were collected by filtration 1 to provide 360 mg of the crystals. Also, by allowing to stand the mother liquor at room temperature, 160 mg oE the prisms were obtained.
In addition, in the case of forming crystals of ;~
- DE-3936, needles or fine prisms were obtained when the crystalli-zation was carried out in a short period of time, while the large prisms were obtained when the crystallization was carried out gradually.
1 9 ,:, -Preparation of the free acid of DE-3936: ;
In 100 ml of acetone was added 1 g of DE-3936 obtained in Example 3 and then 50 ml of water was added to the solution, pH of which was about 7.2. After adjusting the pH of the solu-tion to 2.9 with 0.5 normal hydrochloric acid, the product was extracted three times with 500 ml of benzene. The extract was washed with water, dehydrated with sodium sulfate, and concentrated to dryness to give the 1 g of white powder. The powder was dissolved in 8 ml of benzene, and the solution was concentrated to about 2 milliliters, and after adding 6 ml of petroleum ; ether thereto, the mixture was allowed to stand at room tem-perature for 2 days, whereby colorless prisms were formed.
The crystals were recovered by filtration and washed with petroleum ether to provide 800 mg of the colorless prisms of DE-3936.
Preparation of the sodium salt of DE~3936~
In 50 ml of acetone was dissolved 1 g of the powder - of free E-3936 obtained in Example S and then 25 ml of water was added to the solution. After adjusting the pH of the solution to 12.2 by adding 0.5 normal aqueous potassium hydroxide solution, .. . , . . . : . : ..
`` ~064420 acetone was distilled off. The residual aqueous solution was extracted with ethyl acetate and the extract was washed with water, dehydrated with sodium sulfate,and concentrated to dryness to provide about 90 mg of white powder. In 10 ml of benzene - was dissolved S00 mg of the powder thus obtained and the solution was concentrated to about 1 milliliter. Then, 5 ml of petroleum ether was added to the concentrate and the mixture was allowed to stand at room temperature, whereby colourless prisms were precipitated. By collecting the crystals, 390 mg of the potassium salt of DE-3936 was obtained.
:",' ` ' ': :
Claims (20)
1. A process for the production of an antibiotic of the formula I
(I) which comprises cultivating a strain of Streptomyces hygro-scopicus No. 9735-1 under aerobic conditions in an aqueous nutrient medium and recovering the antibiotic from the medium
(I) which comprises cultivating a strain of Streptomyces hygro-scopicus No. 9735-1 under aerobic conditions in an aqueous nutrient medium and recovering the antibiotic from the medium
2. The process as claimed in claim 1 wherein the carbon source of said aqueous nutrient medium is selected from the group consisting of glucose, fructose, sucrose, dextrin, starch, molasses and glycerol.
3. The process as claimed in claim 2 where the carbon source is glucose.
4. The process as claimed in claim 1 where the nitrogen source of said aqueous nutrient medium is selected from the group consisting of soybean meal, cotton seed flour, distiller's soluble, corn steep liquor, yeast, yeast extract and polypeptone.
5. The process as claimed in claim 1 wherein said aqueous nutrient medium further includes one or more materials selected from the group consisting of a fatty acid or an ester or salt thereof, inorganic salts and an anti-foaming agent.
6. The process as claimed in claim 1 wherein the pH of the medium is from 6.0 to 7.5.
7. The process as claimed in claim 1 wherein the temperature of the process is from 26°C to 31°C.
8. The process as claimed in claim 1 wherein the accum-mulation of the cultivation occurs from 65 to 110 hours.
9. The process as claimed in claim 1 wherein said medium is shorter during cultivation.
10. The process as claimed in claim 1 wherein said cultivation is an aerobic submerged cultivation.
11. The process as claimed in claim 10 wherein said cultivation is conducted in a fermentation tank.
12. The process as claimed in claim 11 wherein said recovery comprises extracting the antibiotic from the fermen-tation broth with an organic solvent, then purifying said extracted antibiotic.
13. The process as claimed in claim 12 wherein said purification is by chromatography or recrystallization.
14. The process as claimed in claim 12 wherein said extraction is carried out subsequent to adjusting the pH of the fermentation broth to from 7.0 to 8Ø
15. The antibiotic of the general formula I of claim 1 wherein prepared by the process as claimed in claims 1, 11 and 12.
16. The process as claimed in claim 1 further including the steps of converting the thus obtained antibiotic to a salt selected from the group consisting of a sodium salt, a potassium salt and an ammonium salt.
17. The process as claimed in claim 16 wherein the free acid of the antibiotic is dissolved in acetone, an aqueous solution of sodium hydroxide is added thereby adjusting the pH
17. The process as claimed in claim 16 wherein the free acid of the antibiotic is dissolved in acetone, an aqueous solution of sodium hydroxide is added thereby adjusting the pH
Claim 17 continued...
to from 9 to 12, and the corresponding sodium salt is extracted and concentrated.
to from 9 to 12, and the corresponding sodium salt is extracted and concentrated.
18. The antibiotic of the following formula II
(II) whenever prepared by the process as claimed in claim 17.
(II) whenever prepared by the process as claimed in claim 17.
19. The potassium salt of the antibiotic of the general formula I of claim 1 whenever prepared by the process as claimed in claim 16.
20. The ammonium salt of the antibiotic of the general formula I of claim 1 whenever prepared by the process as claimed in claim 16.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP50089536 | 1975-07-22 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1064420A true CA1064420A (en) | 1979-10-16 |
Family
ID=13973525
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA257,348A Expired CA1064420A (en) | 1975-07-22 | 1976-07-20 | Antiprotozoal agent for animals |
Country Status (10)
| Country | Link |
|---|---|
| AR (1) | AR209976A1 (en) |
| AT (1) | AT349139B (en) |
| BG (1) | BG28270A3 (en) |
| CA (1) | CA1064420A (en) |
| ES (1) | ES450428A1 (en) |
| IT (1) | IT1062633B (en) |
| MX (1) | MX5831E (en) |
| NZ (1) | NZ181531A (en) |
| PH (1) | PH14840A (en) |
| ZA (1) | ZA764157B (en) |
-
1976
- 1976-07-13 ZA ZA764157A patent/ZA764157B/en unknown
- 1976-07-19 PH PH18699A patent/PH14840A/en unknown
- 1976-07-20 CA CA257,348A patent/CA1064420A/en not_active Expired
- 1976-07-20 BG BG7633789A patent/BG28270A3/en unknown
- 1976-07-21 NZ NZ181531A patent/NZ181531A/en unknown
- 1976-07-21 MX MX761790U patent/MX5831E/en unknown
- 1976-07-21 IT IT5053376A patent/IT1062633B/en active
- 1976-07-22 ES ES450428A patent/ES450428A1/en not_active Expired
- 1976-07-22 AT AT538776A patent/AT349139B/en not_active IP Right Cessation
- 1976-07-22 AR AR264045A patent/AR209976A1/en active
Also Published As
| Publication number | Publication date |
|---|---|
| NZ181531A (en) | 1978-04-28 |
| IT1062633B (en) | 1984-10-20 |
| ATA538776A (en) | 1978-08-15 |
| ES450428A1 (en) | 1977-08-16 |
| AT349139B (en) | 1979-03-26 |
| BG28270A3 (en) | 1980-03-25 |
| ZA764157B (en) | 1977-06-29 |
| MX5831E (en) | 1984-07-31 |
| AR209976A1 (en) | 1977-06-15 |
| PH14840A (en) | 1981-12-16 |
| AU1610576A (en) | 1977-06-16 |
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