CA1080145A - Antibiotic a-30912 mixture from aspergillus ragulosas - Google Patents
Antibiotic a-30912 mixture from aspergillus ragulosasInfo
- Publication number
- CA1080145A CA1080145A CA261,528A CA261528A CA1080145A CA 1080145 A CA1080145 A CA 1080145A CA 261528 A CA261528 A CA 261528A CA 1080145 A CA1080145 A CA 1080145A
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- antibiotic
- factor
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- culture medium
- factors
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/50—Cyclic peptides containing at least one abnormal peptide link
- C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
- C07K7/56—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring the cyclisation not occurring through 2,4-diamino-butanoic acid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Pharmacology & Pharmacy (AREA)
- General Chemical & Material Sciences (AREA)
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- Proteomics, Peptides & Aminoacids (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Compounds Of Unknown Constitution (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
ABSTRACT
This invention relates to a noval antibiotic A-3012 mixture comprising antibiotic A-30912 factors A, B, C, D, E, F and G. It also relates to a novel process for the production of antibiotic A-30912 mixture by submerged aerobic fermentation by Aspergillus rugulosus NRRL 8113, separation of antibiotic A-30912 mixture from the culture medium and the isolation of antibiotic A-30912 factors A, B, C, D, E, F and G from the antibiotic A-30912 mixture. Anti-biotic A-30912 mixture and antibiotic A-30912 factors A, B, C, D, E, F and G are antifungal agents.
This invention relates to a noval antibiotic A-3012 mixture comprising antibiotic A-30912 factors A, B, C, D, E, F and G. It also relates to a novel process for the production of antibiotic A-30912 mixture by submerged aerobic fermentation by Aspergillus rugulosus NRRL 8113, separation of antibiotic A-30912 mixture from the culture medium and the isolation of antibiotic A-30912 factors A, B, C, D, E, F and G from the antibiotic A-30912 mixture. Anti-biotic A-30912 mixture and antibiotic A-30912 factors A, B, C, D, E, F and G are antifungal agents.
Description
1084~145 This invention relates to a novel antibiotic A-30912 mixture comprising at least 7 individual factors A, B, C, D, E, F and G, The antibiotic A-30912 mixture is produced by culturing a novel strain of the organism Aspergillus ru~ulosus NRRL 8113.
The term "antibiotic mixture" as used in this -;:
specification refers to a mixture of co-produced individual antibiotic factors. As will be recognized by those familiar with antibiotic production by fermentation, the ratio of 10 individual factors produced in an antibiotic mixture will .
vary, depending on the fermentation conditions used.
The individual antibiotic factors of the present :
invention are designated antibiotic A-30912 factors A, B, C, ..
D, E, F, and G. ;
It is the object of this invention to provide the novel antibiotic A-30912 mixture and antibiotic A-30912 factors B, C, D, E, F and G.
It is a].so the object of this invention to provide processes for procluction and separation o antibiotic A-30912 mixture comprising factors A, B, C, D, E, F and G
and the isolation of factors A, B, C, D, E, F and G.
The present invention provides a novel antibiotic A-30912 mixture comprising factors A, B, C, D, E, F and G. ;, The present invention also provides the novel process for production of antibiotic A-30912 mixture com-prising factors A, B, C, D, E, F and G comprising:
a) cultivation of Asper~lllus rugulosus NRRL 8113 in a culture medium containing assimilable sources of carbo-.
101~145 hydrate, nitrogen, and inorganic salts under submerged aerobic fermentation conditions until a substantial amount of antibiotic activity is produced; and b) the separation of the antibiotic A-30912 mixture from the culture medium; and c) optionally, the isolation of antibiotic A-30912 factors A, B, C, D, E, F or G from the antibiotic A-30912 mixture.
The antibiotic A-30912 mixture is extracted from the fermentation medium with polar organic solvents.
The known compound sterigmatocystin is also pro-duced by Aspergillus rugulosus NRRL 8113. Sterigmatocystin is extracted either separately with a nonpolar organic solvent or together with the antibiotic A-30912 mixture with polar organic solvents. In the latter case, the antibiotic A-30912 mixture is separated from sterigmato-cystin by concentrating the extracting solvent, adding the concentrate to an excess of a nonpolar organic solvent such as diethyl ether, and separating the A-30912 antibiotic mix-ture as a precipitate. Sterigmatocystin is separated in thefiltrate. The antibiotic A-30912 mixture is further puri-fied by column chromatography.
The antibiotic A-30912 mixture and the individual A-30912 factors are antifungal agents.
. :~ . . ' ' , . :
108014~i Infrared absorption spectra of the following A-30912 factors in KBr disc are presented in the accom-panying drawings:
Figure 1 - antibiotic A-30912 factor A
Figure 2 - antibiotic A-30912 factor D
Figure 3 - antibiotic A-30912 factor B -Figure 4 - antibiotic A-30912 factor C
Antibiotic A-30912 factor A
A-30912 factor A is also similar to the polypep-tide antibiotic Echinocandin B recently reported by F. Benz et al., Helv. Chim. Acta_57, 2459-2477 (1974).
Antibiotic A-30912 factor A is a white amorphous solid. Elemental analysis of A-30912 factor A gave the following percentage composition:
Carbon, 56.52%; hydrogen, 7.29%; nitrogen, 8.68%; -oxygen, 27.09%.
The approximate empirical formula proposed for antibiotic A-30912 factor A i8 C51_53H79-83N717-19 approximate range, the elemental analysis of A-30912 factor A corresponds especially well with an empirical formula of C52H81N7O18-H2O (Calcd.: C, 56.24; H, 7.54; N, 8-84;
O, 27.39).
Antibiotic A-30912 factor A has an approximate molecular weight of 1100, as determined by mass spectrometry and titration.
The infrared absorption spectrum of antibiotic A-30912 factor A in KBr disc is shown in figure 1 of the accompanying drawinqs. The following characteristic absorp-1~801~5 tion maxima are observed: 2.97 (strong), 3.39 (medium), 3.47 (weak), 5.99 (strong), 6.10 (Strohg), 6.49 (medium), 6.56 (medium), 6.90 (medium), 8.00 (weak), 9.13 (weak), and 11.77 (weak) microns.
The ultraviolet absorption spectra of antibiotic A-30912 factor A in both neutral and acidic methanol exhibit absorption maxima at 225 nm (~ 18,000), 275 nm (~ 3,000) and 284 nm (shoulder ~ 2,500). The ultraviolet spectrum of factor A in basic methanol shows absorption maxima at 245 nm ~ 16,000) and 290 nm (~ 3,000) and also end absorp-tion.
The 13C nuclear magnetic resonance spectrum of antibiotic A-30912 factor A in perdeuteromethanol shows the following characteristics:
~ 176.1, 174.3, 173.4, 172.7, 172.4, 169.~, 158.4, 132.8, 130.9, 129.6, 129.0, 116.2, 77.0, 75.7, 74.4, 71.3, 70.9, 69.6, 68.3, 62.4, 58.7, 56.9, 56.1, 52.9, 39.0, 38.5, 36.8, 35.2, 33.9, 32.9, 32.6, 30.7, 30.4, 30.2, 28.2, 27.0, 26.5, 23.6, 20.1, 19.6, 14.4, and 11.3 ppm.
Antibiotic A-30912 factor A has the following specific rotations: [a]D ~44 (c 0.5, CH3OH) [a]3565 -156 (c 0.5, CH30H) Electrometric titration of antibiotic A-30912 factor A in 66% aqueous dimethylformamide indicated the presence of a titratable group with a PKa value of 12.8 (initial pH 6.9).
Amino-acid analysis of antibiotic A-30912 factor A
indicated the presence, after hydrolysis, of threonine, . : . .
.
108~45 hydroxyproline and three other as-yet-unidentified amino acids.
Antibiotic A-30912 factor A is soluble in a variety of organic solvents such as methanol, ethanol, dimethylformamide, dimethyl sulfoxide, and ethyl acetate;
but is insoluble in nonpolar organic solvents such as diethyl ether and petroleum ether. Antibiotic A-30912 factor A is also soluble in aqueous solutions, especially those having a pH greater than 7Ø
A-3091? Factor D
Antibiotic A-30912 factor D is a white amorphous solid. Elemental analysis of A-30912 factor D gave the following percentage composition: carbon, 56.37 percent;
hydrogen, 8.17 percent; nitrogen, 8.54 percent; oxygen (by difference), 26.92 percent.
Antibiotic A-3091Z factor D has an approximate molecular weight of 1100, based upon amino-acid analysis and its close structural relationship to antibiotic A-30912 factor A.
The infrared absorption spectrum of antibiotic A-30912 factor D in KBr disc is shown in figure 2 of the accompanying drawings. The following characteristic absorp-tion maxima are observed: 2.98 (strong), 3.31 (weak), 3.36 (shoulder), 3.40 (medium), 3.48 (weak), 5.76 (weak), 6.01 (strong), 6.10 (shoulder), 6.49 (medium), 6.57 (medium), 6.90 (medium), 7.81 (weak), 8.07 (weak), and 9.16 (weak) microns.
The ultraviolet absorption (UV) spectra of anti-biotic A-30912 factor D in neutral and acidic methanol X-4~46~ -6-8V14~
exhibit absorption maxima at 225 nm (~ 18,000) and 275 nm ( 2,500). The UV spectrum of A-30912 factor D in basic methanol exhibits absorption maxima at 240 nm ( 11,000) and 290 nm (~ 3,000).
Antibiotic A~30912 factor D has the following specific rotation: [~]D ~50 (c 0.34, CH30H).
Amino-acid analysis of antibiotic A-30912 factor D, after hydrolysis, indicated the presence of threonine, hydroxyproline, histidine and three other as-yet-unidenti-fied amino acids. One of the unidentified antibiotic A-30912-factor-D amino acids is identical to one of the unidentified antibiotic A-30912-factor-A amino acids. ~;
Antibiotic A-30912 factor D is soluble in a variety of organic solvents such as methanol, ethanol, dimethylformamide, dimethyl sulfoxide, and ethyl acetate;
but is insoluble in non-polar organic solvents such as diethyl ether and petroleum ether. Antibiotic A-30912 factor D is soluble in aqueous solutions, especially those having a pH greater than 7Ø
A-30912 Factor B
Antibiotic A-30912 factor B is a white amorphous solid. Elemental analysis of A-30912 factor B gave the following approximate percentage composition: carbon, 57.36 percent; hydrogen, 5.92 percent; nitrogen, 8.75 percent; oxygen, 26.19 percent.
The infrared absorption spectrum of A-30912 ractor B in KBr disc is shown in figure 3 of the accom-~)any;ng drawings. The following characteristic absorption maxima are observed: 2.99, 3.41, 3.49, 6.06, 6.15, 6.54, 30 6.61, 6.94, 7.62, 8.07, 9.26, and 9.39 microns.
lasv~s The ultraviolet absorption spectra of A-30912 factor B in both neutral and acidic methanol exhibit ab-sorption maxima at 223 nm (shoulder, 16,000) and 278 nm (~ ;~
The term "antibiotic mixture" as used in this -;:
specification refers to a mixture of co-produced individual antibiotic factors. As will be recognized by those familiar with antibiotic production by fermentation, the ratio of 10 individual factors produced in an antibiotic mixture will .
vary, depending on the fermentation conditions used.
The individual antibiotic factors of the present :
invention are designated antibiotic A-30912 factors A, B, C, ..
D, E, F, and G. ;
It is the object of this invention to provide the novel antibiotic A-30912 mixture and antibiotic A-30912 factors B, C, D, E, F and G.
It is a].so the object of this invention to provide processes for procluction and separation o antibiotic A-30912 mixture comprising factors A, B, C, D, E, F and G
and the isolation of factors A, B, C, D, E, F and G.
The present invention provides a novel antibiotic A-30912 mixture comprising factors A, B, C, D, E, F and G. ;, The present invention also provides the novel process for production of antibiotic A-30912 mixture com-prising factors A, B, C, D, E, F and G comprising:
a) cultivation of Asper~lllus rugulosus NRRL 8113 in a culture medium containing assimilable sources of carbo-.
101~145 hydrate, nitrogen, and inorganic salts under submerged aerobic fermentation conditions until a substantial amount of antibiotic activity is produced; and b) the separation of the antibiotic A-30912 mixture from the culture medium; and c) optionally, the isolation of antibiotic A-30912 factors A, B, C, D, E, F or G from the antibiotic A-30912 mixture.
The antibiotic A-30912 mixture is extracted from the fermentation medium with polar organic solvents.
The known compound sterigmatocystin is also pro-duced by Aspergillus rugulosus NRRL 8113. Sterigmatocystin is extracted either separately with a nonpolar organic solvent or together with the antibiotic A-30912 mixture with polar organic solvents. In the latter case, the antibiotic A-30912 mixture is separated from sterigmato-cystin by concentrating the extracting solvent, adding the concentrate to an excess of a nonpolar organic solvent such as diethyl ether, and separating the A-30912 antibiotic mix-ture as a precipitate. Sterigmatocystin is separated in thefiltrate. The antibiotic A-30912 mixture is further puri-fied by column chromatography.
The antibiotic A-30912 mixture and the individual A-30912 factors are antifungal agents.
. :~ . . ' ' , . :
108014~i Infrared absorption spectra of the following A-30912 factors in KBr disc are presented in the accom-panying drawings:
Figure 1 - antibiotic A-30912 factor A
Figure 2 - antibiotic A-30912 factor D
Figure 3 - antibiotic A-30912 factor B -Figure 4 - antibiotic A-30912 factor C
Antibiotic A-30912 factor A
A-30912 factor A is also similar to the polypep-tide antibiotic Echinocandin B recently reported by F. Benz et al., Helv. Chim. Acta_57, 2459-2477 (1974).
Antibiotic A-30912 factor A is a white amorphous solid. Elemental analysis of A-30912 factor A gave the following percentage composition:
Carbon, 56.52%; hydrogen, 7.29%; nitrogen, 8.68%; -oxygen, 27.09%.
The approximate empirical formula proposed for antibiotic A-30912 factor A i8 C51_53H79-83N717-19 approximate range, the elemental analysis of A-30912 factor A corresponds especially well with an empirical formula of C52H81N7O18-H2O (Calcd.: C, 56.24; H, 7.54; N, 8-84;
O, 27.39).
Antibiotic A-30912 factor A has an approximate molecular weight of 1100, as determined by mass spectrometry and titration.
The infrared absorption spectrum of antibiotic A-30912 factor A in KBr disc is shown in figure 1 of the accompanying drawinqs. The following characteristic absorp-1~801~5 tion maxima are observed: 2.97 (strong), 3.39 (medium), 3.47 (weak), 5.99 (strong), 6.10 (Strohg), 6.49 (medium), 6.56 (medium), 6.90 (medium), 8.00 (weak), 9.13 (weak), and 11.77 (weak) microns.
The ultraviolet absorption spectra of antibiotic A-30912 factor A in both neutral and acidic methanol exhibit absorption maxima at 225 nm (~ 18,000), 275 nm (~ 3,000) and 284 nm (shoulder ~ 2,500). The ultraviolet spectrum of factor A in basic methanol shows absorption maxima at 245 nm ~ 16,000) and 290 nm (~ 3,000) and also end absorp-tion.
The 13C nuclear magnetic resonance spectrum of antibiotic A-30912 factor A in perdeuteromethanol shows the following characteristics:
~ 176.1, 174.3, 173.4, 172.7, 172.4, 169.~, 158.4, 132.8, 130.9, 129.6, 129.0, 116.2, 77.0, 75.7, 74.4, 71.3, 70.9, 69.6, 68.3, 62.4, 58.7, 56.9, 56.1, 52.9, 39.0, 38.5, 36.8, 35.2, 33.9, 32.9, 32.6, 30.7, 30.4, 30.2, 28.2, 27.0, 26.5, 23.6, 20.1, 19.6, 14.4, and 11.3 ppm.
Antibiotic A-30912 factor A has the following specific rotations: [a]D ~44 (c 0.5, CH3OH) [a]3565 -156 (c 0.5, CH30H) Electrometric titration of antibiotic A-30912 factor A in 66% aqueous dimethylformamide indicated the presence of a titratable group with a PKa value of 12.8 (initial pH 6.9).
Amino-acid analysis of antibiotic A-30912 factor A
indicated the presence, after hydrolysis, of threonine, . : . .
.
108~45 hydroxyproline and three other as-yet-unidentified amino acids.
Antibiotic A-30912 factor A is soluble in a variety of organic solvents such as methanol, ethanol, dimethylformamide, dimethyl sulfoxide, and ethyl acetate;
but is insoluble in nonpolar organic solvents such as diethyl ether and petroleum ether. Antibiotic A-30912 factor A is also soluble in aqueous solutions, especially those having a pH greater than 7Ø
A-3091? Factor D
Antibiotic A-30912 factor D is a white amorphous solid. Elemental analysis of A-30912 factor D gave the following percentage composition: carbon, 56.37 percent;
hydrogen, 8.17 percent; nitrogen, 8.54 percent; oxygen (by difference), 26.92 percent.
Antibiotic A-3091Z factor D has an approximate molecular weight of 1100, based upon amino-acid analysis and its close structural relationship to antibiotic A-30912 factor A.
The infrared absorption spectrum of antibiotic A-30912 factor D in KBr disc is shown in figure 2 of the accompanying drawings. The following characteristic absorp-tion maxima are observed: 2.98 (strong), 3.31 (weak), 3.36 (shoulder), 3.40 (medium), 3.48 (weak), 5.76 (weak), 6.01 (strong), 6.10 (shoulder), 6.49 (medium), 6.57 (medium), 6.90 (medium), 7.81 (weak), 8.07 (weak), and 9.16 (weak) microns.
The ultraviolet absorption (UV) spectra of anti-biotic A-30912 factor D in neutral and acidic methanol X-4~46~ -6-8V14~
exhibit absorption maxima at 225 nm (~ 18,000) and 275 nm ( 2,500). The UV spectrum of A-30912 factor D in basic methanol exhibits absorption maxima at 240 nm ( 11,000) and 290 nm (~ 3,000).
Antibiotic A~30912 factor D has the following specific rotation: [~]D ~50 (c 0.34, CH30H).
Amino-acid analysis of antibiotic A-30912 factor D, after hydrolysis, indicated the presence of threonine, hydroxyproline, histidine and three other as-yet-unidenti-fied amino acids. One of the unidentified antibiotic A-30912-factor-D amino acids is identical to one of the unidentified antibiotic A-30912-factor-A amino acids. ~;
Antibiotic A-30912 factor D is soluble in a variety of organic solvents such as methanol, ethanol, dimethylformamide, dimethyl sulfoxide, and ethyl acetate;
but is insoluble in non-polar organic solvents such as diethyl ether and petroleum ether. Antibiotic A-30912 factor D is soluble in aqueous solutions, especially those having a pH greater than 7Ø
A-30912 Factor B
Antibiotic A-30912 factor B is a white amorphous solid. Elemental analysis of A-30912 factor B gave the following approximate percentage composition: carbon, 57.36 percent; hydrogen, 5.92 percent; nitrogen, 8.75 percent; oxygen, 26.19 percent.
The infrared absorption spectrum of A-30912 ractor B in KBr disc is shown in figure 3 of the accom-~)any;ng drawings. The following characteristic absorption maxima are observed: 2.99, 3.41, 3.49, 6.06, 6.15, 6.54, 30 6.61, 6.94, 7.62, 8.07, 9.26, and 9.39 microns.
lasv~s The ultraviolet absorption spectra of A-30912 factor B in both neutral and acidic methanol exhibit ab-sorption maxima at 223 nm (shoulder, 16,000) and 278 nm (~ ;~
2,400). The ultraviolet spectrum of antibiotic A-30912 factor B in basic methanol shows absorption maxima at 242 nm (~ 13,900) and 292 nm (~ 2,800).
A-30912 factor B has the following approximate specific rotations: [a]D -47 (c 0.5, CH30H) [a]365 -170 (c 0.5, CH30H).
Electrometric titration of A-30912 factor B in 66 aqueous dimethylformamide indicated the presence of a titratable group with a PKa value of about 13.0 (initial pH
7.91).
Amino-acid analysis of A-30912 factor B indicated the presence, after standard acid hydrolysis, of threonine, hydroxyproline and several as-yet-unidentified amino acids.
A-30912 factor B is soluble in a variety of organic solvents such as methanol, ethanol, dimethylformamide, dimethyl sulfoxide, and ethyl acetate; but is insoluble in nonpolar organic solvents such as diethyl ether and petroleum ether. A-30912 factor B is also soluble in aqueous solutions, especially those having a pH greater than 7Ø
A-30912 Factor C
Antibiotic A-30912 factor C is a white amorphous solid. Elemental analysis of A-30912 factor C gave the following approximate percentage composition: carbon, 56.76 percent; hydrogen, 7.88 percent; nitrogen, 10.61 percent;
oxygen, 25.09 percent.
The infrared absorption spectrum of A-30912 factor C in KBr disc is shown in figure 4 of the accompanying drawings. The following characteristic absorption maxima -are observed: 2.98, 3.39, 3.43, 3.51, 6.01, 6 12, 6.47, 6.90, 7.04, 7.22, 7.38, 8.00, 8.30, and 9.13 microns.
The ultraviolet absorption spectra of A-30912 factor C in both neutral and acidic methanol exhibit ab-sorption maxima at 223 nm (shoulder, 7,300) and 275 nm (E
1,350). The ultraviolet spectrum of antibiotic A-30912 factor C in basic methanol shows absorption maxima at 240 nm (~ 12,400) and 290 nm ( 5,200).
A-30912 factor C has the following approximate specific rotations: []D5 ~33 (c 0.5, CH30H) [a]365 -119 (c 0.5, CH30H).
Electrometric titration of A-30912 factor C in 66%
aqueous dimethylformamide indicated the presence of a titratable group with a PKa value of about 13.08 (initial pH
7.93).
Amino-acid analysis of A-30912 factor C indicated the presence, after standard acid hydrolysis, of threonine, hydroxyproline and several as-yet-unidentified amino acids.
A-30912 factor C is soluble in a variety of organic solvents such as methanol, ethanol, dimethylformamide, dimethyl sulfoxide, and ethyl acetate; but is insoluble in nonpolar organic solvents such as diethyl ether and petroleum ether. A-30912 factor C is also soluble in aqueous solutions, especially those having a pH greater than 7Ø
1~80145 The seven individual factors of the antibiotic A-30912 mixture can be separated and identifièd by the use of thin-layer chromatography (TLC). Silica gel is a pre-ferred adsorbent; and benzene:methanol (7:3, V:V) is a preferred solvent system.
The Rf values of antibiotic A-30912 factors A-G, using silica gel (Merck, Darmstadt) TLC, the benzene:meth-anol (7:3) solvent system, and Candida albicans bioauto- .
graphy are given in Table I.
Antibiotic A-30912 Factor Rf Value ,~
A 0.35 B 0,45 C 0.54 D 0.59 ~ :
E 0.27 F 0.18 G 0.13 The Rf values of antibiotic A-30912 factor A in various paper-chromatographic systems, again using Candida albicans as a detection organism, are given in Table II.
X-4646~ -10-TABLE II
A-30912 Factor A
Rf Value Solvent system 0.76 sutanol saturated with water 0.69 Butanol saturated with water plus 2% p-toluenesulfonic acid 0.75 Methanol:0.1 N HCl (3:1) 0.17 Butanol:ethanol:water (13.5:15:150) 0.78 Methanol:0.05 M sodium citrate at pH 5.7 (7:3); paper buff-ered with 0.05 M sodium ci-trate at pH 5.7 ~ ' The organism useful for the preparation of the ; antibiotic A-30912 mixture was isolated from a soil sample from the ruins of Pompeii, Italy. The A-30912 producing organism is classified as a strain of Aspergillus rugulosus Thom and Raper, which is in the Asper~illus nidulans form group. This classification is based on the description of K. B. Raper and D. I. Fennel in "The Genus Aspergillus,"
The Williams and Wilkins Company, Baltimore, Md., 1965.
Color names were assigned according to the ISCC-NBS method (K. L. Kelly and D. B. Judd, "The ISCC-NBS Method of Designating Color and a Dictionary of Color Names," U. S.
Dept. of Commerce, Circ. 553, Washington, D. C., 1955). The Maerz and Paul color blocks are described by A. Maerz and M.
R. Paul in "Dictionary of Color," McGraw-Hill Book Company, New York, N.Y., I950.
Cultures were grown at 25 C. unless otherwise specified.
iO80145 Culture Characteristics of A. rugulosus NRRL 8113 -Cza~ek's Solution ~ r ~ -~ . .
The culture grows slowly, attaining 1.5 to 2.0 cm in diameter in 15 days at 25C. The colony surface is -`?
convex and velutinous, becoming wrinkled with age near the center and then umbonate. The mycelium periphery is a-2-mm wide band of deeply submerged colorless hyphae and is sinuate. A pinkish-brown exudate forms on the marginal aerial hyphae. In from 7 to 14 days a pale purple soluble pigment is produced in the agar surrounding the colony. The pigment diffuses throughout the colony by 15 days.
After 5 days the colony surface ranges from white to buff, and the colony reverse is brownish orange centrally and brownish to brownish purple in the peripheral regions.
In 10 days the colony is moderate yellowish pink (ISCC-NBS
29 and Maerz and Paul ll-A-7). After 14 days the colony is light grayish red (ISCC-NBS 18 and Maera and Paul 4-G-7).
The marginal area becomes verruculose and is strong yellow (ISCC-NBS 84 and Maerz and Paul 10-L-5) due to conidiation.
Scattered dull yellowish clusters of hulle cells occur randomly over the surface and along the margin of the colony. With age, the strong yellow patches and marginal area become yellowish green. After 3 weeks, an orange-purple tone is observed in the new aerial components of the margin. Initially, the colony reverse is slightly concave.
~s it matures, the colony flattens to the agar surface, and ~he reverse becomes slightly wrinkled. By 10 days the reverse is light brown (ISCC-NBS 57 and Maerz and Paul 5-A-10). In 15 days it is grayish red (ISCC-NBS 19 and Maerz and Paul 6-J-3).
.
:
The conidiogenous state is sparse; and conidio-phores are scattered over the surface, sometimes occurring as patches or in a submarginal band. Conidial heads are at first loosely radiate and globose; with aging, they may develop as short columnar forms which are more compact.
Globose heads are from 70~ to 80~ in diameter and average 50~. Columnar heads may be up to 140~ long and 70~ wide.
Conidia are globose to subglobose, rugulose, and greenish gold en masse. They range from 2.8~ to 3.9~ in diameter and average 3.2~.
Sterigmata are biseriate and colorless. Primary sterigmata range in length from 4.7~ to 11.0~ and average 7.9lJ- At their widest point they are 2.4~ and taper to 1.6~. Secondary sterigmata may occur singly or in pairs, arising from the primaries, and are flask shaped. At their widest point they measure 3.0~ and taper apically to 0.4~ -where they become tubular. The tubular apex is 1.2~ long.
The overall length range~ from 5.5~ to 12.6~ and averages 9.21J.
Vesicles are globose to subglobose or hemispheri-cal and may be apically flattened, becoming brownish with age. They range from 7.4~ to 11.2~ in diameter and average 9.4ll.
Conidiophores are smooth, relatively thick-walled, and are at first hyaline then evolve to a light cinnamon brown. They are slightly wider at the vesicle and may taper slightly near the foot cell. The average width is 5.9~l.
X-464hA -13-~801~5 ~ ~
Conidiophores range from 47.7~ to 166.6~ and average 106~ in length. They arise from submerged hyphae or laterally from aerial hyphal filaments.
The ascogenous state appears in up to 20 days.
The small yellowish clusters of hulle cells which occur on the surface may be found at any level in the mycelium. They consist of hulle cells which envelope one or more cleisto-thecia. Hulle cells are globose to subglobose or oval to elongate, are thick-walled and hyaline. Globose hulle cells range from 18~ to 24~ in diameter and average 21.8~.
Cleistothecia are globose to subglobose, thick-walled, relatively tough and fibrous. At first relatively colorless, they become reddish purple and darken with age.
They measure from 165~ to 4701l in diameter and average 275~.
Malt Extract Agar Colonies grown at 25 C. expand rapidly, attaining 4-5 cm in 10-12 days. At first a grayish white, colonies become moderate olive green (ISCC-NBS ~0 and Maerz and Paul 23-E-6) in 4 days. The sinuate to weakly lobate periphery consists of tightly packed, short, white aerial hyphae.
Small yellowish clusters of hulle cells dot the margin and are randomly scattered over the felt-like agar surface.
After 20 days these hulle cell clusters tend to encrust much of the surface. The colony reverse is grayish yellow (ISCC-NBS 90 and Maerz and Paul ll-B-l).
The ascogenous state appears in 15 days. The small yellowish clusters of hulle cells which occur on the surface may be found at any level in the mycelium. They consist of hulle cells which envelope one or more cleisto-X-4646~ -14-1080~45 thecia. Hulle cells may encrust large areas over the conidial heads. Hulle cells are globose to subglobose or oval to elongate, are thick-walled and hyaline. Globose hulle cells range from 18~ to 24~ in diameter and average 21.8~. ;
Cleistothecia are globose to subglobose and are dark reddish brown. They range from 389~ to 700~ in diam-eter and average 506~.
Asci are fragile, hyaline, and subglobose to oval.
Subglobose asci are from 8.7~ to 11.9~ in diameter and average 10.3~. Oval asci are from 10.3~-14.2~ x 8.7~-10.31l and average 12.2~ x 9.1~.
Ascospores are red-orange, rugulose, with two parallel, delicately pleated equatorial crests which are up to 0.8~ wide and unbroken. The ascospore appears lenticular through the long axis. When the crest is peripheral, the ascospore is globose. In the globose view it is from 4.9 to 6.3~ in diameter and averages 5.4~.
__ Two characteristics of the antibiotic A-30912-producing strain of Aspergillus rugulosus differ from the characteristics of A. rugulosus described by Raper and Fennel, supra. The A-30912-producing strain has larger conidial heads and ascospores.
The Aspergillus rugulosus culture useful for the prod~lction of the antibiotic A-30912 mixture has been deposited and made a part oL the stock culture collection of the Northern Regional Research Laboratory, U. S. Department .
~ )145 of ~griculture, Agricultural Research Service, Peoria, Illinois 61604, from which it is available to the public under the number NRRL 8113.
Any one of a number of culture medium can be employed to grow Aspergillus rugulosus NRRL 8113. For economy in production, optimal yield and ease of product isolation, however, certain culture media are preferred.
Thus, for example, a preferred carbohydrate source in large-scale fermentation is glucose, although molasses, starch, lactose, sucrose, maltose, glycerol and the like may be employed. Preferred nitrogen sources are enzyme-hydrolyzed casein and soluble meat peptone, although distiller's grains, monosodium glutamate and the like may be used. Nutrient inorganic salts can be incorporated in the culture media.
These include the customary soluble salts capable of yielding sodium, magnesium, calcium, ammonium, chloride, carbonate, sulfate, nitrate, and the like ions.
Essential trace elements necessary for the growth and development of the organism should also be included in the culture medium. Such trace elements commonly occur as impurities in other constituents of the medium in amounts sufficient to meet the growth requirements of the organism.
It may be necessary to add small amounts (i.e.
0.2 ml./l.) of an antifoam agent such as polypropylene c~lycol to large-scale fermentation media if foaming becomes a ~roblem.
Ior ~roduction of a substantial quantity of the anti~ioti( ~-~0912 mixturc, submerged aerobic fermentation ',. ..
1~1)145 in tanks is preferred. Small quantities of the antibiotic A-30912 mixture may be obtained by shake-flask culture.
secause of the time lag in antibiotic production commonly associated with inoculation of large tanks with the spore form of the organism, it is preferable to use a vegetative inoculum. The vegetative inoculum is prepared by inocu-lating a small volume of culture medium with the spore form or mycelial fragments of the organism to obtain a fresh, actively growing culture of the organism. The vegetative inoculum is then transferred to a larger tank. The medium used for the growth of the vegetative inoculum can be the same as that used for larger fermenf:ations, but other media can also be employed. The antibiotic A-30912-producing organism can be grown at temperatures between about 20 and about 43C.; the organism grows well at temperatures of about 25-30C. Optimum production of the antibiotic A-30912 mixture appears to occur at a temperature of about 25C.
As is customary in aerobic submerged culture processes, sterile air is blown through the culture medium.
For efficient antibiotic production, the volume of air employed in tank production is preferably above 0.4 volume of air per volume of culture medium per minute (V/V/M).
Production of the antibiotic A-30912 mixture can be followed during the fermentation by testing samples of alcoholic extracts of the whole broth for antibiotic activity against an organism known to be sensitive to the A-30912 antibiotics. One assay organism useful in testing for the l)resence of the antibiotic A-30912 mixture is Candida albicans.
'rhe bioassay is conveniently performed by paper-disc assay on seeded agar plates.
11~80145 Generally, antibiotic activity can be detected on the second day of fermentation. Maximum production of antibiotic activity usually occurs between about the third and the sixth days.
The antibiotic A-30912 factors are antifungal agents. Illustrative of the antifungal activity of the -antibiotic A-30912 factors are ln vitro tests with anti-biotic A-30912 factors A and D. These tests are summarized in Table III. Antifungal activity was measured by the conventional disc-diffusion method (6-mm pads were dipped in solutions containing test compound; pads were placed on agar -plates seeded with the test organism). Results are given as the minimal inhibitory concentration (MIC) per disc at which the test compound inhibited the test organism.
TABLE III
Test Organism MIC (mcg/disc) Antibiotic Antibiotic Factor A Factor D
Candida albicans 0.625 0.5 -Trichophyton mentagrophytes 0.078 0.07 A-30912 factor A is very active in in ln vitro disc-diffusion tests against dermatophytes. The results of these tests are summarized in Table IV.
; . . .:
- 108l)145 ~:
TABLE IV
A-30912 FACTOR A VS. DERMATOPHYTES
Dermatophyte No. of Isolates MIC~mcg/disc) TrichophrYton mentagrophytes13 1.25 - .039 ,,:
Trichophyton gallinae 1 >1.25 :~
Trichophyton :' me~inini 1 . 0 0195 Trichophyton quinckeanum 1 >1 25 :
-:
10 TrichophYton rubrum 1 <.0098 ~ .
Trichophyton schoencelnii 1 0.0195 Trichophyton terrestre 1 0.0195 Trichophyton tonsuranrs- ~ 9 >1.25 - 0.156 Microsporium -gypseum 5 0.156 - 0.038 Microsporiu~
audouinii 4 1.25 - 0.156 Microsporium canis~ ~ - 6 1.25 - 0.0098 20 Microsporium cookel 2 1.25 - 0.0195 .
Nannizzia incurvata 1 0.312 : . .
Phalaphere jean SAlemi 1 >l. 25 ~
Epidermatophyton floccosum 1 1.25 .
Geotrichum candidum~~~ 4 ~1.25 - 0.156 Keratinomyces ajelllo 1 0.156 .. ..
10~V145 The antifungal activity of the antibiotic A-30912 factors was further demonstrated by in vivo tests. The in vivo tests were carried out in the following manner: Three doses of test compound are given to Candida albicans-infected mice at 0, 4, and 24 hours post-infection. The protection which the test compound provides is measured as an ED50 value [the effective dose in mg/kg which protects 50 percent of the mice; see W. Wick et al., J. Bacteriol. 81, 233-235 tl961) 1. The ED50 values for antibiotic A-30912 factor A against Candida albicans in mice were 30 mg/kg (intraperitoneal administration) and 50 mg/kg(subcutaneous administration). The ED50 value for antibiotic A-30912 factor D against Candida albicans in mice was 33 mg/kg (subcutaneous administration).
There were no signs of acute toxicity when anti-biotic A-30912 factor A was administered intraperitoneally (ip) or subcutaneously (sc) to mice at 100 mg/kg twice per day for three days (a total of 600 mg/kg). There were also no signs of acute toxicity when antibiotic A-30912 factor A
20 was administered ip to mice at 200 mg/kg three ti~es per day ( a total dose of 600 mg/kg).
There were no signs of acute toxicity when anti-biotic A-30912 factor D was administered sc to mice three times per day at 14 mg/kg (a total dose of 42 mg/kg).
When used as antifungal agents, the antibiotic A-30912 factors are administered parenterally and are com-m~nly administered together with a pharmaceutically-accep-table carrier or diluent. The dosage of antibiotic A-30912 factor will depend upon a variety of conditions, such as the nature and severity of the particular infection involved.
In order to illustrate more fully the operation of this invention, the following examples are provided.
A. Shake-flask Fermentation A culture of Aspergillus rugulosus NRRL 8113 was prepared and maintained on an 18- x 150- ml agar slant having the following composition:
Amount Ingredient (percent) Dextrin 1.0000 Enzymatic hydrolysate of casein* 0.2000 Yeast extract 0.1000 Beef extract 0.1000 ~;
KCl 0.0200 MgSO ~7H2O 0.0200 FeS04 7H20 Water 98.5596 *"N-Z-Amine A,"l Sheffield Chemical Co., Norwich, N.Y.
The slant was inoculated with Aspergillus rugu-losus NRRL 8113, and the inoculated slant was incubated at 25C. for about 7 days. The mature slant culture was covered with beef serum and scraped with a sterile loop to loosen the spores. One-half of the resulting suspension was used to inoculate 50 ml of a vegetative medium having the following composition:
1 Trademark '~ ~ . . .
108()145 Amount Ingredient (Percent) Sucrose 2.5 Molasses 3.6 Corn steep liquor 0.6 Enzymatic hydrolysate of casein* 1.0 K2HPO4 0.2 Water 92.1 *"N-Z-Case"2,Sheffield Chemical Co., Norwich, N.Y.
The inoculated vegetative medium was incubated in ;~
a 250-ml wide-mouth Erlenmeyer flask at 25~ C. for 24 hours on a shaker rotating through an arc two inches in diameter at 250 RPM.
This incubated vegetative medium may be used directly to inoculate the second-stage vegetative medium.
Alternatively and preferably, it can be stored for later use by maintaining the culture in the vapor phase of liquid nitrogen. The culture is prepared for such storage in multiple small vials as follows: In each vial is placed 2 ml of incubclted vegetative medium and 2 ml of a glycerol-lactose solution having the following composition: , Ingredient Amount Glycerol 20%
Lactose 10%
Deionized water 70~
Thc prcpared suspensions are stored in the vapor phase of liquid nitrogen.
2. Trademark ~r~
.
- 1080~45 A stored suspension (1 ml) thus prepared was used to inoculate 50 ml of a first-stage vegetative medium having the same composition earlier described for the vegetative medium. The inoculated first-stage vegetative medium was incubated in a 250-ml wide-mouth Erlenmeyer flask at 25 C
for 22 hours on a shaker rotating through an arc two inches in diameter at 250 RPM.
B. Tank Fermentation In order to provide a larger volume of inoculum, 10 ml of the above-described incubated first-stage vege-tative medium was used to inoculate 400 ml of a second-stage vegetative growth medium having the same composition as that of the vegetative medium. The second-stage medium was incubated in a 2-liter wide-mouth Erlenmeyer flask at 25 C for 25 hours on a shaker rotating through an arc two `
inches in diameter at 250 RPM. ~;
Incubated second-stage vegetative medium (800 ml), prepared as above-described, was used to inoculate 100 liters of sterile production medium having the following composition:
Ingredient Amount Glucose 25 g/liter Starch 10 g/liter Peptone* 10 g/liter Blackstrap molasses 5 g/liter Enzymatic hydrolysate of casein** 4 g/liter g O4 712O 0.5 g/liter Czapek's mineral stock*** 2.0 ml/liter CaCO3 2.0 g/liter Deionized water q.s. 1 liter . . .
1081)145 *W.P. No. 159, Inolex Biomedical Corp., Glenwood. Ill.
**"N-Z Amine A", Sheffield Chemical Co., Norwich, N.Y.
***Czapek's mineral stock has the following composition: `
Ingredient Amount FeSO4.7H2O (dissolved in 2 ml conc HCl) 2 g KCl 100 g MgSO4.7H2O 100 g Deionized water q.s. to 1 liter The pH of the medium was 6.8 after sterilization by autoclaving at 121C for 30 min at about 16-18 pounds pressure. The inoculated production medium was allowed to ferment in a 165-liter fermentation tank at a temperature of 25C for four days. The fermentation medium was aerated with sterile air at the rate of 0.5 V/V/M. The fermentation med-ium was stirred with conventional agitators at 300 RPM.
Separation of the Antibiotic A-30912 Mixture Whole fermentation broth (200 1.), obtained by the method described in Example 1, was stirred thoroughly with methanol (200 1.) for one hour and then was filtered, using `
a filter aid ("Hyflo Super-cel"3, a diatomaceous earth, Johns-Manville Products Corp.). The pH of the filtrate was ad- `
justed to pH 4.0 by the addition of 5 N HCl. The acidified filtrate was extracted twice with equal volumes of chloro- i~
form. The chloroform extracts were combined and concentrated
A-30912 factor B has the following approximate specific rotations: [a]D -47 (c 0.5, CH30H) [a]365 -170 (c 0.5, CH30H).
Electrometric titration of A-30912 factor B in 66 aqueous dimethylformamide indicated the presence of a titratable group with a PKa value of about 13.0 (initial pH
7.91).
Amino-acid analysis of A-30912 factor B indicated the presence, after standard acid hydrolysis, of threonine, hydroxyproline and several as-yet-unidentified amino acids.
A-30912 factor B is soluble in a variety of organic solvents such as methanol, ethanol, dimethylformamide, dimethyl sulfoxide, and ethyl acetate; but is insoluble in nonpolar organic solvents such as diethyl ether and petroleum ether. A-30912 factor B is also soluble in aqueous solutions, especially those having a pH greater than 7Ø
A-30912 Factor C
Antibiotic A-30912 factor C is a white amorphous solid. Elemental analysis of A-30912 factor C gave the following approximate percentage composition: carbon, 56.76 percent; hydrogen, 7.88 percent; nitrogen, 10.61 percent;
oxygen, 25.09 percent.
The infrared absorption spectrum of A-30912 factor C in KBr disc is shown in figure 4 of the accompanying drawings. The following characteristic absorption maxima -are observed: 2.98, 3.39, 3.43, 3.51, 6.01, 6 12, 6.47, 6.90, 7.04, 7.22, 7.38, 8.00, 8.30, and 9.13 microns.
The ultraviolet absorption spectra of A-30912 factor C in both neutral and acidic methanol exhibit ab-sorption maxima at 223 nm (shoulder, 7,300) and 275 nm (E
1,350). The ultraviolet spectrum of antibiotic A-30912 factor C in basic methanol shows absorption maxima at 240 nm (~ 12,400) and 290 nm ( 5,200).
A-30912 factor C has the following approximate specific rotations: []D5 ~33 (c 0.5, CH30H) [a]365 -119 (c 0.5, CH30H).
Electrometric titration of A-30912 factor C in 66%
aqueous dimethylformamide indicated the presence of a titratable group with a PKa value of about 13.08 (initial pH
7.93).
Amino-acid analysis of A-30912 factor C indicated the presence, after standard acid hydrolysis, of threonine, hydroxyproline and several as-yet-unidentified amino acids.
A-30912 factor C is soluble in a variety of organic solvents such as methanol, ethanol, dimethylformamide, dimethyl sulfoxide, and ethyl acetate; but is insoluble in nonpolar organic solvents such as diethyl ether and petroleum ether. A-30912 factor C is also soluble in aqueous solutions, especially those having a pH greater than 7Ø
1~80145 The seven individual factors of the antibiotic A-30912 mixture can be separated and identifièd by the use of thin-layer chromatography (TLC). Silica gel is a pre-ferred adsorbent; and benzene:methanol (7:3, V:V) is a preferred solvent system.
The Rf values of antibiotic A-30912 factors A-G, using silica gel (Merck, Darmstadt) TLC, the benzene:meth-anol (7:3) solvent system, and Candida albicans bioauto- .
graphy are given in Table I.
Antibiotic A-30912 Factor Rf Value ,~
A 0.35 B 0,45 C 0.54 D 0.59 ~ :
E 0.27 F 0.18 G 0.13 The Rf values of antibiotic A-30912 factor A in various paper-chromatographic systems, again using Candida albicans as a detection organism, are given in Table II.
X-4646~ -10-TABLE II
A-30912 Factor A
Rf Value Solvent system 0.76 sutanol saturated with water 0.69 Butanol saturated with water plus 2% p-toluenesulfonic acid 0.75 Methanol:0.1 N HCl (3:1) 0.17 Butanol:ethanol:water (13.5:15:150) 0.78 Methanol:0.05 M sodium citrate at pH 5.7 (7:3); paper buff-ered with 0.05 M sodium ci-trate at pH 5.7 ~ ' The organism useful for the preparation of the ; antibiotic A-30912 mixture was isolated from a soil sample from the ruins of Pompeii, Italy. The A-30912 producing organism is classified as a strain of Aspergillus rugulosus Thom and Raper, which is in the Asper~illus nidulans form group. This classification is based on the description of K. B. Raper and D. I. Fennel in "The Genus Aspergillus,"
The Williams and Wilkins Company, Baltimore, Md., 1965.
Color names were assigned according to the ISCC-NBS method (K. L. Kelly and D. B. Judd, "The ISCC-NBS Method of Designating Color and a Dictionary of Color Names," U. S.
Dept. of Commerce, Circ. 553, Washington, D. C., 1955). The Maerz and Paul color blocks are described by A. Maerz and M.
R. Paul in "Dictionary of Color," McGraw-Hill Book Company, New York, N.Y., I950.
Cultures were grown at 25 C. unless otherwise specified.
iO80145 Culture Characteristics of A. rugulosus NRRL 8113 -Cza~ek's Solution ~ r ~ -~ . .
The culture grows slowly, attaining 1.5 to 2.0 cm in diameter in 15 days at 25C. The colony surface is -`?
convex and velutinous, becoming wrinkled with age near the center and then umbonate. The mycelium periphery is a-2-mm wide band of deeply submerged colorless hyphae and is sinuate. A pinkish-brown exudate forms on the marginal aerial hyphae. In from 7 to 14 days a pale purple soluble pigment is produced in the agar surrounding the colony. The pigment diffuses throughout the colony by 15 days.
After 5 days the colony surface ranges from white to buff, and the colony reverse is brownish orange centrally and brownish to brownish purple in the peripheral regions.
In 10 days the colony is moderate yellowish pink (ISCC-NBS
29 and Maerz and Paul ll-A-7). After 14 days the colony is light grayish red (ISCC-NBS 18 and Maera and Paul 4-G-7).
The marginal area becomes verruculose and is strong yellow (ISCC-NBS 84 and Maerz and Paul 10-L-5) due to conidiation.
Scattered dull yellowish clusters of hulle cells occur randomly over the surface and along the margin of the colony. With age, the strong yellow patches and marginal area become yellowish green. After 3 weeks, an orange-purple tone is observed in the new aerial components of the margin. Initially, the colony reverse is slightly concave.
~s it matures, the colony flattens to the agar surface, and ~he reverse becomes slightly wrinkled. By 10 days the reverse is light brown (ISCC-NBS 57 and Maerz and Paul 5-A-10). In 15 days it is grayish red (ISCC-NBS 19 and Maerz and Paul 6-J-3).
.
:
The conidiogenous state is sparse; and conidio-phores are scattered over the surface, sometimes occurring as patches or in a submarginal band. Conidial heads are at first loosely radiate and globose; with aging, they may develop as short columnar forms which are more compact.
Globose heads are from 70~ to 80~ in diameter and average 50~. Columnar heads may be up to 140~ long and 70~ wide.
Conidia are globose to subglobose, rugulose, and greenish gold en masse. They range from 2.8~ to 3.9~ in diameter and average 3.2~.
Sterigmata are biseriate and colorless. Primary sterigmata range in length from 4.7~ to 11.0~ and average 7.9lJ- At their widest point they are 2.4~ and taper to 1.6~. Secondary sterigmata may occur singly or in pairs, arising from the primaries, and are flask shaped. At their widest point they measure 3.0~ and taper apically to 0.4~ -where they become tubular. The tubular apex is 1.2~ long.
The overall length range~ from 5.5~ to 12.6~ and averages 9.21J.
Vesicles are globose to subglobose or hemispheri-cal and may be apically flattened, becoming brownish with age. They range from 7.4~ to 11.2~ in diameter and average 9.4ll.
Conidiophores are smooth, relatively thick-walled, and are at first hyaline then evolve to a light cinnamon brown. They are slightly wider at the vesicle and may taper slightly near the foot cell. The average width is 5.9~l.
X-464hA -13-~801~5 ~ ~
Conidiophores range from 47.7~ to 166.6~ and average 106~ in length. They arise from submerged hyphae or laterally from aerial hyphal filaments.
The ascogenous state appears in up to 20 days.
The small yellowish clusters of hulle cells which occur on the surface may be found at any level in the mycelium. They consist of hulle cells which envelope one or more cleisto-thecia. Hulle cells are globose to subglobose or oval to elongate, are thick-walled and hyaline. Globose hulle cells range from 18~ to 24~ in diameter and average 21.8~.
Cleistothecia are globose to subglobose, thick-walled, relatively tough and fibrous. At first relatively colorless, they become reddish purple and darken with age.
They measure from 165~ to 4701l in diameter and average 275~.
Malt Extract Agar Colonies grown at 25 C. expand rapidly, attaining 4-5 cm in 10-12 days. At first a grayish white, colonies become moderate olive green (ISCC-NBS ~0 and Maerz and Paul 23-E-6) in 4 days. The sinuate to weakly lobate periphery consists of tightly packed, short, white aerial hyphae.
Small yellowish clusters of hulle cells dot the margin and are randomly scattered over the felt-like agar surface.
After 20 days these hulle cell clusters tend to encrust much of the surface. The colony reverse is grayish yellow (ISCC-NBS 90 and Maerz and Paul ll-B-l).
The ascogenous state appears in 15 days. The small yellowish clusters of hulle cells which occur on the surface may be found at any level in the mycelium. They consist of hulle cells which envelope one or more cleisto-X-4646~ -14-1080~45 thecia. Hulle cells may encrust large areas over the conidial heads. Hulle cells are globose to subglobose or oval to elongate, are thick-walled and hyaline. Globose hulle cells range from 18~ to 24~ in diameter and average 21.8~. ;
Cleistothecia are globose to subglobose and are dark reddish brown. They range from 389~ to 700~ in diam-eter and average 506~.
Asci are fragile, hyaline, and subglobose to oval.
Subglobose asci are from 8.7~ to 11.9~ in diameter and average 10.3~. Oval asci are from 10.3~-14.2~ x 8.7~-10.31l and average 12.2~ x 9.1~.
Ascospores are red-orange, rugulose, with two parallel, delicately pleated equatorial crests which are up to 0.8~ wide and unbroken. The ascospore appears lenticular through the long axis. When the crest is peripheral, the ascospore is globose. In the globose view it is from 4.9 to 6.3~ in diameter and averages 5.4~.
__ Two characteristics of the antibiotic A-30912-producing strain of Aspergillus rugulosus differ from the characteristics of A. rugulosus described by Raper and Fennel, supra. The A-30912-producing strain has larger conidial heads and ascospores.
The Aspergillus rugulosus culture useful for the prod~lction of the antibiotic A-30912 mixture has been deposited and made a part oL the stock culture collection of the Northern Regional Research Laboratory, U. S. Department .
~ )145 of ~griculture, Agricultural Research Service, Peoria, Illinois 61604, from which it is available to the public under the number NRRL 8113.
Any one of a number of culture medium can be employed to grow Aspergillus rugulosus NRRL 8113. For economy in production, optimal yield and ease of product isolation, however, certain culture media are preferred.
Thus, for example, a preferred carbohydrate source in large-scale fermentation is glucose, although molasses, starch, lactose, sucrose, maltose, glycerol and the like may be employed. Preferred nitrogen sources are enzyme-hydrolyzed casein and soluble meat peptone, although distiller's grains, monosodium glutamate and the like may be used. Nutrient inorganic salts can be incorporated in the culture media.
These include the customary soluble salts capable of yielding sodium, magnesium, calcium, ammonium, chloride, carbonate, sulfate, nitrate, and the like ions.
Essential trace elements necessary for the growth and development of the organism should also be included in the culture medium. Such trace elements commonly occur as impurities in other constituents of the medium in amounts sufficient to meet the growth requirements of the organism.
It may be necessary to add small amounts (i.e.
0.2 ml./l.) of an antifoam agent such as polypropylene c~lycol to large-scale fermentation media if foaming becomes a ~roblem.
Ior ~roduction of a substantial quantity of the anti~ioti( ~-~0912 mixturc, submerged aerobic fermentation ',. ..
1~1)145 in tanks is preferred. Small quantities of the antibiotic A-30912 mixture may be obtained by shake-flask culture.
secause of the time lag in antibiotic production commonly associated with inoculation of large tanks with the spore form of the organism, it is preferable to use a vegetative inoculum. The vegetative inoculum is prepared by inocu-lating a small volume of culture medium with the spore form or mycelial fragments of the organism to obtain a fresh, actively growing culture of the organism. The vegetative inoculum is then transferred to a larger tank. The medium used for the growth of the vegetative inoculum can be the same as that used for larger fermenf:ations, but other media can also be employed. The antibiotic A-30912-producing organism can be grown at temperatures between about 20 and about 43C.; the organism grows well at temperatures of about 25-30C. Optimum production of the antibiotic A-30912 mixture appears to occur at a temperature of about 25C.
As is customary in aerobic submerged culture processes, sterile air is blown through the culture medium.
For efficient antibiotic production, the volume of air employed in tank production is preferably above 0.4 volume of air per volume of culture medium per minute (V/V/M).
Production of the antibiotic A-30912 mixture can be followed during the fermentation by testing samples of alcoholic extracts of the whole broth for antibiotic activity against an organism known to be sensitive to the A-30912 antibiotics. One assay organism useful in testing for the l)resence of the antibiotic A-30912 mixture is Candida albicans.
'rhe bioassay is conveniently performed by paper-disc assay on seeded agar plates.
11~80145 Generally, antibiotic activity can be detected on the second day of fermentation. Maximum production of antibiotic activity usually occurs between about the third and the sixth days.
The antibiotic A-30912 factors are antifungal agents. Illustrative of the antifungal activity of the -antibiotic A-30912 factors are ln vitro tests with anti-biotic A-30912 factors A and D. These tests are summarized in Table III. Antifungal activity was measured by the conventional disc-diffusion method (6-mm pads were dipped in solutions containing test compound; pads were placed on agar -plates seeded with the test organism). Results are given as the minimal inhibitory concentration (MIC) per disc at which the test compound inhibited the test organism.
TABLE III
Test Organism MIC (mcg/disc) Antibiotic Antibiotic Factor A Factor D
Candida albicans 0.625 0.5 -Trichophyton mentagrophytes 0.078 0.07 A-30912 factor A is very active in in ln vitro disc-diffusion tests against dermatophytes. The results of these tests are summarized in Table IV.
; . . .:
- 108l)145 ~:
TABLE IV
A-30912 FACTOR A VS. DERMATOPHYTES
Dermatophyte No. of Isolates MIC~mcg/disc) TrichophrYton mentagrophytes13 1.25 - .039 ,,:
Trichophyton gallinae 1 >1.25 :~
Trichophyton :' me~inini 1 . 0 0195 Trichophyton quinckeanum 1 >1 25 :
-:
10 TrichophYton rubrum 1 <.0098 ~ .
Trichophyton schoencelnii 1 0.0195 Trichophyton terrestre 1 0.0195 Trichophyton tonsuranrs- ~ 9 >1.25 - 0.156 Microsporium -gypseum 5 0.156 - 0.038 Microsporiu~
audouinii 4 1.25 - 0.156 Microsporium canis~ ~ - 6 1.25 - 0.0098 20 Microsporium cookel 2 1.25 - 0.0195 .
Nannizzia incurvata 1 0.312 : . .
Phalaphere jean SAlemi 1 >l. 25 ~
Epidermatophyton floccosum 1 1.25 .
Geotrichum candidum~~~ 4 ~1.25 - 0.156 Keratinomyces ajelllo 1 0.156 .. ..
10~V145 The antifungal activity of the antibiotic A-30912 factors was further demonstrated by in vivo tests. The in vivo tests were carried out in the following manner: Three doses of test compound are given to Candida albicans-infected mice at 0, 4, and 24 hours post-infection. The protection which the test compound provides is measured as an ED50 value [the effective dose in mg/kg which protects 50 percent of the mice; see W. Wick et al., J. Bacteriol. 81, 233-235 tl961) 1. The ED50 values for antibiotic A-30912 factor A against Candida albicans in mice were 30 mg/kg (intraperitoneal administration) and 50 mg/kg(subcutaneous administration). The ED50 value for antibiotic A-30912 factor D against Candida albicans in mice was 33 mg/kg (subcutaneous administration).
There were no signs of acute toxicity when anti-biotic A-30912 factor A was administered intraperitoneally (ip) or subcutaneously (sc) to mice at 100 mg/kg twice per day for three days (a total of 600 mg/kg). There were also no signs of acute toxicity when antibiotic A-30912 factor A
20 was administered ip to mice at 200 mg/kg three ti~es per day ( a total dose of 600 mg/kg).
There were no signs of acute toxicity when anti-biotic A-30912 factor D was administered sc to mice three times per day at 14 mg/kg (a total dose of 42 mg/kg).
When used as antifungal agents, the antibiotic A-30912 factors are administered parenterally and are com-m~nly administered together with a pharmaceutically-accep-table carrier or diluent. The dosage of antibiotic A-30912 factor will depend upon a variety of conditions, such as the nature and severity of the particular infection involved.
In order to illustrate more fully the operation of this invention, the following examples are provided.
A. Shake-flask Fermentation A culture of Aspergillus rugulosus NRRL 8113 was prepared and maintained on an 18- x 150- ml agar slant having the following composition:
Amount Ingredient (percent) Dextrin 1.0000 Enzymatic hydrolysate of casein* 0.2000 Yeast extract 0.1000 Beef extract 0.1000 ~;
KCl 0.0200 MgSO ~7H2O 0.0200 FeS04 7H20 Water 98.5596 *"N-Z-Amine A,"l Sheffield Chemical Co., Norwich, N.Y.
The slant was inoculated with Aspergillus rugu-losus NRRL 8113, and the inoculated slant was incubated at 25C. for about 7 days. The mature slant culture was covered with beef serum and scraped with a sterile loop to loosen the spores. One-half of the resulting suspension was used to inoculate 50 ml of a vegetative medium having the following composition:
1 Trademark '~ ~ . . .
108()145 Amount Ingredient (Percent) Sucrose 2.5 Molasses 3.6 Corn steep liquor 0.6 Enzymatic hydrolysate of casein* 1.0 K2HPO4 0.2 Water 92.1 *"N-Z-Case"2,Sheffield Chemical Co., Norwich, N.Y.
The inoculated vegetative medium was incubated in ;~
a 250-ml wide-mouth Erlenmeyer flask at 25~ C. for 24 hours on a shaker rotating through an arc two inches in diameter at 250 RPM.
This incubated vegetative medium may be used directly to inoculate the second-stage vegetative medium.
Alternatively and preferably, it can be stored for later use by maintaining the culture in the vapor phase of liquid nitrogen. The culture is prepared for such storage in multiple small vials as follows: In each vial is placed 2 ml of incubclted vegetative medium and 2 ml of a glycerol-lactose solution having the following composition: , Ingredient Amount Glycerol 20%
Lactose 10%
Deionized water 70~
Thc prcpared suspensions are stored in the vapor phase of liquid nitrogen.
2. Trademark ~r~
.
- 1080~45 A stored suspension (1 ml) thus prepared was used to inoculate 50 ml of a first-stage vegetative medium having the same composition earlier described for the vegetative medium. The inoculated first-stage vegetative medium was incubated in a 250-ml wide-mouth Erlenmeyer flask at 25 C
for 22 hours on a shaker rotating through an arc two inches in diameter at 250 RPM.
B. Tank Fermentation In order to provide a larger volume of inoculum, 10 ml of the above-described incubated first-stage vege-tative medium was used to inoculate 400 ml of a second-stage vegetative growth medium having the same composition as that of the vegetative medium. The second-stage medium was incubated in a 2-liter wide-mouth Erlenmeyer flask at 25 C for 25 hours on a shaker rotating through an arc two `
inches in diameter at 250 RPM. ~;
Incubated second-stage vegetative medium (800 ml), prepared as above-described, was used to inoculate 100 liters of sterile production medium having the following composition:
Ingredient Amount Glucose 25 g/liter Starch 10 g/liter Peptone* 10 g/liter Blackstrap molasses 5 g/liter Enzymatic hydrolysate of casein** 4 g/liter g O4 712O 0.5 g/liter Czapek's mineral stock*** 2.0 ml/liter CaCO3 2.0 g/liter Deionized water q.s. 1 liter . . .
1081)145 *W.P. No. 159, Inolex Biomedical Corp., Glenwood. Ill.
**"N-Z Amine A", Sheffield Chemical Co., Norwich, N.Y.
***Czapek's mineral stock has the following composition: `
Ingredient Amount FeSO4.7H2O (dissolved in 2 ml conc HCl) 2 g KCl 100 g MgSO4.7H2O 100 g Deionized water q.s. to 1 liter The pH of the medium was 6.8 after sterilization by autoclaving at 121C for 30 min at about 16-18 pounds pressure. The inoculated production medium was allowed to ferment in a 165-liter fermentation tank at a temperature of 25C for four days. The fermentation medium was aerated with sterile air at the rate of 0.5 V/V/M. The fermentation med-ium was stirred with conventional agitators at 300 RPM.
Separation of the Antibiotic A-30912 Mixture Whole fermentation broth (200 1.), obtained by the method described in Example 1, was stirred thoroughly with methanol (200 1.) for one hour and then was filtered, using `
a filter aid ("Hyflo Super-cel"3, a diatomaceous earth, Johns-Manville Products Corp.). The pH of the filtrate was ad- `
justed to pH 4.0 by the addition of 5 N HCl. The acidified filtrate was extracted twice with equal volumes of chloro- i~
form. The chloroform extracts were combined and concentrated
3. Trademark 108~145 under vacuum to a volume of about four liters. This con-centrate was added to about 60 liters of diethyl ether to precipitate the A-30912 mixture. The precipitate was -~
separated by filtration and dried to give 38 g of the antibiotic A-30912 mixture as a gray powder. The filtrate was concentrated under vacuum to give an oil; this oil was dissolved in methanol (500 ml). The methanol solution was added to diethyl ether (7.5 1.) to precipitate additional antibiotic A-30912 mixture. This precipitate was also separated by filtration and dried to give and additional 3.5 g of the antibiotic A-30912 mixture.
Isolation of Antibiotic A-30912 Factor A
, ._ - - :
Antibiotic A-30912 mixture (20 g), obtained as described in Example 2, was placed on a silica-gel column (4- x 107-cm, Woelm) in acetonitrile:water (95:5). The column was eluted with acetonitrile:water (95:5) at a flow rate of 1 to 2 ml per minute, collecting fractions having a volume of approximately 20 ml. Fractions were checked by thin-layer silica-gel chromatography, using the acetoni-trile:water (95:5) solvent system and Candida albicans bioautography.
Fractions 74 through 125 were combined and con-centrated. The concentrated solution crystallized upon standing to give an additional 124 mg of sterigmatocystin.
Fractions 212 through 273 were combined and concentrated under vacuum to give an oil. This oil was dissolved in a small volume of methanol. The methanol solution was added . . :
- - ~: .
10~ 45 ., to diethyl ether (15 volumes). The precipitate which formed ;
was separated and dried to give 23 mg of antibiotic A-30912 factor D. Fractions 274 through 437 contained antibiotic A-30912 factors A, B, C and D. Fractions 482 through 900 contained antibiotic A-30912 factors A, E, F and G. Frac-tions 438 through 481 were combined and concentrated under vacuum to give an oil. This oil was dissolved in a s~all volume of metllanol; and the methanol solution was added to diethyl ether (15 volumes). The precipitate which formed 10 was separated and dried to give 2.17 g of antibiotic A-30912 factor A.
Isolation of Antibiotic A-3091~ Factor D
A partially purified antibiotic A-30912 mixture containing antibiotic A-30912 factors B, C, and D was obtained as described in Example 3 for fractions 274-437.
This material (750 mg) was chromatographed on a silica-gel column (2.2 x 51 cm, Woelm silica gel), collecting fractions having a volume of approximately 15 ml and eluting with the following solvents:
Fractions Solvent .. ..
1-25 acetonitrile 26-62 acetonitrile + 1% water 63-700 acetonitrile + 1.5~ water The column fractions were monitored by silica-ge~l thin-layer chromatography, using acetonitrile:water (95:5) and benzene:methanol (7:3) solvent systems and , ,~ , ", : ~ .: - . ; . .
Candida albicans bioautography. Fractions 535-685, which contained antibiotic A-30912 factor D, were combined and concentrated under vacuum to give an oil. ThiS oil was dissolved in a small amount of methanol and added to diethyl ether (15 volumes~. The precipitate which formed was - ~-separated by filtration and dried to give 69 mg of anti-biotic A-30912 factor D. ;, Isolation of A-30912 factors E~ and C
Partially purified A-30912 antibiotic complex containing A-30912 factors A, B, C, and D was obtained as described in Example 3 for fractions 212-437. This material (18 g) was dissolved in a minimal volume of acetonitrile:
water (4:1) and chromatographed on an aluminum oxide column (3.8 x 56 cm, Woelm), collecting fractions having a volume of approximately 20 ml. The column was eluted with the following solvents:
Fractions Solvent .. . .
1-300 acetonitrile:water (4:1) 301-509 acetonitrile:water (7:3) The column fractions were monitored by silica-gel thin-layer chromatography as described in Example 4. On the basis of these results, fractions were combined and concentrated to oils; the oily residues were dissolved into small volumes of methanol; the methanol solutions were ~recipitated with 10-15 volumes of diethyl ether. The manner in which the fractions were combined, the weight of material obtained, and the factor content of the combined fractions are summarized below.
,. . :
108~)14~;
Fraction Weight (g) Factors 6-28 0.23* --6-28 5.80 A-30912 C, D
34-114 2.90 A-30912 B
115-164 1.20 A-30912 A, B
165-509 1.90 A-30912 A
*insoluble material obtained before ether pre-cipitation In order to obtain purified A-30912 factor C, a portion of fractions 6-28 (2 g) was dissolved in methanol, adsorbed onto a sufficient quantity of silica gel (grade 62), dried, and added on top of a silica-gel column (1.9 x 80 cm, grade 62), packed in acetonitrile. The column was eluted with acetonitrile at a flow rate of 2 ml/minute, collecting factions having a volume of about 10 ml. At fraction 117, the solvent was changed to acetonitrile:water (99:1). The column fractions were again monitored by thin-layer chroma-tography. On the basis of the TLC results, fractions were combined and concentrated to give oily residues; the oily residues were dis601ved into a small volumes of methanol;
the methanol solutions were precipitated with 10-15 volumes of diethyl ether. The factor content and weight of the fractions of interest are summarized below:
Fractions Weight (g) Factors 341-479 0.250 D
480-540 0.015 D
541-899 0.391 C, D
900-1675 0.340 C
: ; ~ , . , , : . . .
separated by filtration and dried to give 38 g of the antibiotic A-30912 mixture as a gray powder. The filtrate was concentrated under vacuum to give an oil; this oil was dissolved in methanol (500 ml). The methanol solution was added to diethyl ether (7.5 1.) to precipitate additional antibiotic A-30912 mixture. This precipitate was also separated by filtration and dried to give and additional 3.5 g of the antibiotic A-30912 mixture.
Isolation of Antibiotic A-30912 Factor A
, ._ - - :
Antibiotic A-30912 mixture (20 g), obtained as described in Example 2, was placed on a silica-gel column (4- x 107-cm, Woelm) in acetonitrile:water (95:5). The column was eluted with acetonitrile:water (95:5) at a flow rate of 1 to 2 ml per minute, collecting fractions having a volume of approximately 20 ml. Fractions were checked by thin-layer silica-gel chromatography, using the acetoni-trile:water (95:5) solvent system and Candida albicans bioautography.
Fractions 74 through 125 were combined and con-centrated. The concentrated solution crystallized upon standing to give an additional 124 mg of sterigmatocystin.
Fractions 212 through 273 were combined and concentrated under vacuum to give an oil. This oil was dissolved in a small volume of methanol. The methanol solution was added . . :
- - ~: .
10~ 45 ., to diethyl ether (15 volumes). The precipitate which formed ;
was separated and dried to give 23 mg of antibiotic A-30912 factor D. Fractions 274 through 437 contained antibiotic A-30912 factors A, B, C and D. Fractions 482 through 900 contained antibiotic A-30912 factors A, E, F and G. Frac-tions 438 through 481 were combined and concentrated under vacuum to give an oil. This oil was dissolved in a s~all volume of metllanol; and the methanol solution was added to diethyl ether (15 volumes). The precipitate which formed 10 was separated and dried to give 2.17 g of antibiotic A-30912 factor A.
Isolation of Antibiotic A-3091~ Factor D
A partially purified antibiotic A-30912 mixture containing antibiotic A-30912 factors B, C, and D was obtained as described in Example 3 for fractions 274-437.
This material (750 mg) was chromatographed on a silica-gel column (2.2 x 51 cm, Woelm silica gel), collecting fractions having a volume of approximately 15 ml and eluting with the following solvents:
Fractions Solvent .. ..
1-25 acetonitrile 26-62 acetonitrile + 1% water 63-700 acetonitrile + 1.5~ water The column fractions were monitored by silica-ge~l thin-layer chromatography, using acetonitrile:water (95:5) and benzene:methanol (7:3) solvent systems and , ,~ , ", : ~ .: - . ; . .
Candida albicans bioautography. Fractions 535-685, which contained antibiotic A-30912 factor D, were combined and concentrated under vacuum to give an oil. ThiS oil was dissolved in a small amount of methanol and added to diethyl ether (15 volumes~. The precipitate which formed was - ~-separated by filtration and dried to give 69 mg of anti-biotic A-30912 factor D. ;, Isolation of A-30912 factors E~ and C
Partially purified A-30912 antibiotic complex containing A-30912 factors A, B, C, and D was obtained as described in Example 3 for fractions 212-437. This material (18 g) was dissolved in a minimal volume of acetonitrile:
water (4:1) and chromatographed on an aluminum oxide column (3.8 x 56 cm, Woelm), collecting fractions having a volume of approximately 20 ml. The column was eluted with the following solvents:
Fractions Solvent .. . .
1-300 acetonitrile:water (4:1) 301-509 acetonitrile:water (7:3) The column fractions were monitored by silica-gel thin-layer chromatography as described in Example 4. On the basis of these results, fractions were combined and concentrated to oils; the oily residues were dissolved into small volumes of methanol; the methanol solutions were ~recipitated with 10-15 volumes of diethyl ether. The manner in which the fractions were combined, the weight of material obtained, and the factor content of the combined fractions are summarized below.
,. . :
108~)14~;
Fraction Weight (g) Factors 6-28 0.23* --6-28 5.80 A-30912 C, D
34-114 2.90 A-30912 B
115-164 1.20 A-30912 A, B
165-509 1.90 A-30912 A
*insoluble material obtained before ether pre-cipitation In order to obtain purified A-30912 factor C, a portion of fractions 6-28 (2 g) was dissolved in methanol, adsorbed onto a sufficient quantity of silica gel (grade 62), dried, and added on top of a silica-gel column (1.9 x 80 cm, grade 62), packed in acetonitrile. The column was eluted with acetonitrile at a flow rate of 2 ml/minute, collecting factions having a volume of about 10 ml. At fraction 117, the solvent was changed to acetonitrile:water (99:1). The column fractions were again monitored by thin-layer chroma-tography. On the basis of the TLC results, fractions were combined and concentrated to give oily residues; the oily residues were dis601ved into a small volumes of methanol;
the methanol solutions were precipitated with 10-15 volumes of diethyl ether. The factor content and weight of the fractions of interest are summarized below:
Fractions Weight (g) Factors 341-479 0.250 D
480-540 0.015 D
541-899 0.391 C, D
900-1675 0.340 C
: ; ~ , . , , : . . .
Claims (17)
1. A process for production of antibiotic A-30912 mixture comprising, factors A, B, C, D, E, F and G
comprising:
a) cultivation of Aspergillus rugulosus NRRL 8113 in a culture medium containing assimilable sources of carbo-hydrate, nitrogen, and inorganic salts under submerged aerobic fermentation conditions until a substantial amount of antibiotic activity is produced; and b) the separation of the antibiotic A-30912 mixture from the culture medium.
comprising:
a) cultivation of Aspergillus rugulosus NRRL 8113 in a culture medium containing assimilable sources of carbo-hydrate, nitrogen, and inorganic salts under submerged aerobic fermentation conditions until a substantial amount of antibiotic activity is produced; and b) the separation of the antibiotic A-30912 mixture from the culture medium.
2. An antibiotic A-30912 mixture comprising factors A, B, C, D, E, F and G, when prepared by the process of claim 1 or by an obvious chemical equivalent thereof.
3. A process as in claim 1, and including the additional step of isolating antibiotic A-30912 factors A, B, C, D, E, F or G from the antibiotic A-30912 mixture.
4. A process for production of antibiotic A-30912 factor A comprising:
a) cultivation of Aspergillus rugulosus NRRL 8113 in a culture medium containing assimilable sources of carbo-hydrate, nitrogen, and inorganic salts under submerged aerobic fermentation conditions until a substantial amount of antibiotic activity is produced;
b) the separation of antibiotic A-30912 mixture from the culture medium; and c) the isolation of antibiotic A-30912 factor A from the antibiotic A-30912 mixture.
a) cultivation of Aspergillus rugulosus NRRL 8113 in a culture medium containing assimilable sources of carbo-hydrate, nitrogen, and inorganic salts under submerged aerobic fermentation conditions until a substantial amount of antibiotic activity is produced;
b) the separation of antibiotic A-30912 mixture from the culture medium; and c) the isolation of antibiotic A-30912 factor A from the antibiotic A-30912 mixture.
5. Antibiotic A-30912 factor A, when prepared by the process of claim 4 or by an obvious chemical equivalent thereof.
6. A process for production of antibiotic A-30912 factor B comprising:
a) cultivation of Aspergillus rugulosus NRRL 8113 in a culture medium containing assimilable sources of carbo-hydrate, nitrogen, and inorganic salts under submerged aerobic fermentation conditions until a substantial amount of antibiotic activity is produced;
b) the separation of antibiotic A-30912 mixture from the culture medium; and c) the isolation of antibiotic A-30912 factor B from the antibiotic A-30912 mixture.
a) cultivation of Aspergillus rugulosus NRRL 8113 in a culture medium containing assimilable sources of carbo-hydrate, nitrogen, and inorganic salts under submerged aerobic fermentation conditions until a substantial amount of antibiotic activity is produced;
b) the separation of antibiotic A-30912 mixture from the culture medium; and c) the isolation of antibiotic A-30912 factor B from the antibiotic A-30912 mixture.
7. Antibiotic A-30912 factor B, when prepared by the process of claim 6 or by an obvious chemical equivalent thereof.
8. A process for production of the antibiotic A-30912 factor C comprising:
a) cultivation of Aspergillus rugulosus NRRL 8113 in a culture medium containing assimilable sources of carbo-hydrate, nitrogen, and inorganic salts under submerged aerobic fermentation conditions until a substantial amount of antibiotic activity is produced;
b) the separation of antibiotic A-30912 mixture from the culture medium; and c) the isolation of antibiotic A-30912 factor C from the antibiotic A-30912 mixture.
a) cultivation of Aspergillus rugulosus NRRL 8113 in a culture medium containing assimilable sources of carbo-hydrate, nitrogen, and inorganic salts under submerged aerobic fermentation conditions until a substantial amount of antibiotic activity is produced;
b) the separation of antibiotic A-30912 mixture from the culture medium; and c) the isolation of antibiotic A-30912 factor C from the antibiotic A-30912 mixture.
9. Antibiotic A-30912 factor C when prepared by the process of claim 8 or by an obvious chemical equivalent thereof.
10. A process for production of the antibiotic A-30912 factor D comprising:
a) cultivation of Aspergillus rugulosus NRRL 8113 in a culture medium containing assimilable sources of carbo-hydrate, nitrogen, and inorganic salts under submerged aerobic fermentation conditions until a substantial amount of antibiotic activity is produced;
b) the separation of antibiotic A-30912 mixture from the culture medium; and c) the isolation of antibiotic A-30912 factor D from the antibiotic A-30912 mixture.
a) cultivation of Aspergillus rugulosus NRRL 8113 in a culture medium containing assimilable sources of carbo-hydrate, nitrogen, and inorganic salts under submerged aerobic fermentation conditions until a substantial amount of antibiotic activity is produced;
b) the separation of antibiotic A-30912 mixture from the culture medium; and c) the isolation of antibiotic A-30912 factor D from the antibiotic A-30912 mixture.
11. Antibiotic A-30912 factor D when prepared by the process of claim 10 or by an obvious chemical equivalent thereof.
12. A process for production of the antibiotic A-30912 factor E comprising:
a) cultivation of Aspergillus rugulosus NRRL 8113 in a culture medium containing assimilable sources of carbo-hydrate, nitrogen, and inorganic salts under submerged aerobic fermentation conditions until a substantial amount of antibiotic activity is produced;
b) the separation of antibiotic A-30912 mixture from the culture medium; and c) the isolation of antibiotic A-30912 factor E from antibiotic A-30912 mixture.
a) cultivation of Aspergillus rugulosus NRRL 8113 in a culture medium containing assimilable sources of carbo-hydrate, nitrogen, and inorganic salts under submerged aerobic fermentation conditions until a substantial amount of antibiotic activity is produced;
b) the separation of antibiotic A-30912 mixture from the culture medium; and c) the isolation of antibiotic A-30912 factor E from antibiotic A-30912 mixture.
13. Antibiotic A-30912 factor E when prepared by the process of claim 12 or by an obvious chemical equivalent thereof.
14. A process for production of the antibiotic A-30912 factor F comprising:
a) cultivation of Aspergillus rugulosus NRRL 8113 in a culture medium containing assimilable sources of carbo-hydrate, nitrogen, and inorganic salts under submerged aerobic fermentation conditions until a substantial amount of antibiotic activity is produced;
b) the separation of antibiotic A-30912 mixture from the culture medium; and c) the isolation of antibiotic A-30912 factor F from the antibiotic A-30912 mixture.
a) cultivation of Aspergillus rugulosus NRRL 8113 in a culture medium containing assimilable sources of carbo-hydrate, nitrogen, and inorganic salts under submerged aerobic fermentation conditions until a substantial amount of antibiotic activity is produced;
b) the separation of antibiotic A-30912 mixture from the culture medium; and c) the isolation of antibiotic A-30912 factor F from the antibiotic A-30912 mixture.
15. Antibiotic A-30912 factor F when prepared by the process of claim 14 or by an obvious chemical equivalent thereof.
16. A process for production of the antibiotic A-30912 factor G comprising:
a) cultivation of Aspergillus rugulosus NRRL 8113 in a culture medium containing assimilable sources of carbo-hydrate, nitrogen, and inorganic salts under submerged aerobic fermentation conditions until a substantial amount of antibiotic activity is produced;
b) the separation of antibiotic A-30912 mixture from the culture medium; and c) the isolation of antibiotic A-30912 factor G from the antibiotic A-30912 mixture.
a) cultivation of Aspergillus rugulosus NRRL 8113 in a culture medium containing assimilable sources of carbo-hydrate, nitrogen, and inorganic salts under submerged aerobic fermentation conditions until a substantial amount of antibiotic activity is produced;
b) the separation of antibiotic A-30912 mixture from the culture medium; and c) the isolation of antibiotic A-30912 factor G from the antibiotic A-30912 mixture.
17. Antibiotic A-30912 factor G when prepared by the process of claim 16 or by an obvious chemical equivalent thereof.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US61910775A | 1975-10-02 | 1975-10-02 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1080145A true CA1080145A (en) | 1980-06-24 |
Family
ID=24480483
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA261,528A Expired CA1080145A (en) | 1975-10-02 | 1976-09-20 | Antibiotic a-30912 mixture from aspergillus ragulosas |
Country Status (28)
| Country | Link |
|---|---|
| JP (1) | JPS5242801A (en) |
| AR (1) | AR209993A1 (en) |
| AT (1) | AT346479B (en) |
| AU (1) | AU506777B2 (en) |
| BE (1) | BE846829A (en) |
| BG (1) | BG25380A3 (en) |
| CA (1) | CA1080145A (en) |
| CH (1) | CH629531A5 (en) |
| CS (1) | CS212776B2 (en) |
| DD (1) | DD127442A5 (en) |
| DE (1) | DE2643485A1 (en) |
| DK (1) | DK143608C (en) |
| ES (1) | ES452021A1 (en) |
| FR (1) | FR2326200A1 (en) |
| GB (2) | GB1499710A (en) |
| GR (1) | GR61730B (en) |
| HU (1) | HU173445B (en) |
| IE (1) | IE43663B1 (en) |
| IL (1) | IL50429A (en) |
| NL (1) | NL7610808A (en) |
| NZ (1) | NZ182123A (en) |
| PL (1) | PL103996B1 (en) |
| PT (1) | PT65617B (en) |
| RO (1) | RO69985A (en) |
| SE (1) | SE7610943L (en) |
| SU (1) | SU620215A3 (en) |
| YU (1) | YU236076A (en) |
| ZA (1) | ZA765320B (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5452797A (en) * | 1977-10-04 | 1979-04-25 | Meiji Seika Kaisha Ltd | Novel antibiotic substance bn-200, and its preparation |
| CA1170598A (en) * | 1979-12-13 | 1984-07-10 | Bernard J. Abbott | Process for the preparation of cyclic peptide nuclei |
| GB2180464A (en) * | 1985-09-18 | 1987-04-01 | British Gas Corp | Gas-solid phase reactions and apparatus therefor |
| FI76004B (en) * | 1986-03-24 | 1988-05-31 | Seppo Kalervo Ruottu | CIRKULATIONSMASSAREAKTOR. |
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1976
- 1976-09-06 IE IE1974/76A patent/IE43663B1/en unknown
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- 1976-09-08 IL IL50429A patent/IL50429A/en unknown
- 1976-09-16 GR GR51709A patent/GR61730B/en unknown
- 1976-09-20 CA CA261,528A patent/CA1080145A/en not_active Expired
- 1976-09-21 NZ NZ182123A patent/NZ182123A/en unknown
- 1976-09-21 PT PT65617A patent/PT65617B/en unknown
- 1976-09-22 JP JP51114357A patent/JPS5242801A/en active Pending
- 1976-09-23 AU AU18080/76A patent/AU506777B2/en not_active Expired
- 1976-09-23 GB GB39477/76A patent/GB1499710A/en not_active Expired
- 1976-09-24 BG BG034273A patent/BG25380A3/en unknown
- 1976-09-27 GB GB39954/76A patent/GB1565821A/en not_active Expired
- 1976-09-27 DE DE19762643485 patent/DE2643485A1/en not_active Withdrawn
- 1976-09-27 YU YU02360/76A patent/YU236076A/en unknown
- 1976-09-29 NL NL7610808A patent/NL7610808A/en not_active Application Discontinuation
- 1976-09-29 CS CS766294A patent/CS212776B2/en unknown
- 1976-09-29 SU SU762404253A patent/SU620215A3/en active
- 1976-09-30 ES ES452021A patent/ES452021A1/en not_active Expired
- 1976-09-30 CH CH1241376A patent/CH629531A5/en not_active IP Right Cessation
- 1976-09-30 DK DK440276A patent/DK143608C/en active
- 1976-10-01 DD DD195092A patent/DD127442A5/xx unknown
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- 1976-10-01 HU HU76EI703A patent/HU173445B/en unknown
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- 1976-10-01 AR AR264953A patent/AR209993A1/en active
- 1976-10-01 SE SE7610943A patent/SE7610943L/en not_active Application Discontinuation
- 1976-10-01 BE BE1007661A patent/BE846829A/en unknown
- 1976-10-01 FR FR7629604A patent/FR2326200A1/en active Granted
- 1976-10-02 RO RO7687891A patent/RO69985A/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| IE43663L (en) | 1977-04-02 |
| IL50429A0 (en) | 1976-11-30 |
| DK143608C (en) | 1982-02-15 |
| AT346479B (en) | 1978-11-10 |
| DK143608B (en) | 1981-09-14 |
| FR2326200A1 (en) | 1977-04-29 |
| GR61730B (en) | 1978-12-30 |
| CH629531A5 (en) | 1982-04-30 |
| HU173445B (en) | 1979-05-28 |
| IL50429A (en) | 1979-11-30 |
| BG25380A3 (en) | 1978-09-15 |
| ZA765320B (en) | 1978-04-26 |
| IE43663B1 (en) | 1981-04-22 |
| BE846829A (en) | 1977-04-01 |
| NZ182123A (en) | 1978-06-02 |
| AU1808076A (en) | 1978-04-06 |
| DE2643485A1 (en) | 1977-04-14 |
| DD127442A5 (en) | 1977-09-21 |
| AU506777B2 (en) | 1980-01-24 |
| ATA730976A (en) | 1978-03-15 |
| CS212776B2 (en) | 1982-03-26 |
| PT65617A (en) | 1976-10-01 |
| PL103996B1 (en) | 1979-07-31 |
| JPS5242801A (en) | 1977-04-04 |
| ES452021A1 (en) | 1977-12-01 |
| GB1499710A (en) | 1978-02-01 |
| DK440276A (en) | 1977-04-03 |
| YU236076A (en) | 1983-04-30 |
| AR209993A1 (en) | 1977-06-15 |
| SU620215A3 (en) | 1978-08-15 |
| RO69985A (en) | 1981-05-15 |
| NL7610808A (en) | 1977-04-05 |
| SE7610943L (en) | 1977-04-03 |
| GB1565821A (en) | 1980-04-23 |
| FR2326200B1 (en) | 1980-03-07 |
| PT65617B (en) | 1978-04-05 |
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