ANIMAL GROWTH PROMOTING AGENTS

Abstract: The invention relates to compositions comprising one or more of the following compounds designated WS-1627A, WS-­ 1627B, WS-1627C and WS-1627D, or salts thereof: wherein R₁ =COCH₃ and R₂=H in WS-1627A (KA-393) and R₁ = H and R₂=COCH₃ in WS-1627C wherein R₁-COCH₃ and R₂=H in WS-1627B (Hygromycin) and R₁=H and R₂=COCH₃ in WS-1627D (Epi-hygromycin) WS-1627A, WS-1627B and WS-1627D are in fact known antibiotics, but WS-1627C is a new epimer of the antibiotic KA-3093. The compositions are useful both for preventing or treat­ ing swine dysentery and as animal growth promotants. There is also provided a process for preparing WS-1672A,

Description: This invention relates to an agent for promoting the growth of growing animals. More particularly, it relates to an animal growth promotant composition which comprises, as effective ingredient(s), one or more compound(s) selected from WS-1627A, WS-1627B, WS-1627C and WS-1627D, or salts thereof. In the course of a screening program for novel prophylactic or therapeutic agents from soil samples, the inventors of the present invention discovered that four substances obtained from a cultured broth of a newly isolated microorganism, Streptomyces noboritoensis subsp. amamiensis subsp. nov. No. 1627, have an animal growth promoting effect. Then, these substances were named WS-1627A, WS-1627B, WS-1627C and WS-1627D, respectively. WS-1627A, WS-1627B, WS-1627C and WS-1627D have the following physicochemical properties:- WS-1627A a) Appearance : White powder b) Melting point : 104-107°C c) Optical rotation : [α] 22 / D = -125.0° (C=1.0, H₂O) d) Molecular weight : 513 [SIMS : m/z 514 (M⁺ + 1)] e) Elemental analysis (%) : C 53.71; H 6.07; N 2.91 f) UV absorption spectrum : 300 (sh, 22800), 270 (34800), 214 (46000) 300 (sh, 22500), 270 (34600), 214 (45900) 323 (sh, 15350), 285 (sh, 25900), 249 (45400), 230 (sh, 41500) g) IR absorption spectrum : 3700-3000, 2930, 1720, 1700, 1640, 1610, 1510, 1420, 1360, 1320, 1270, 1250, 1200, 1130, 1100, 1060, 1025, 960, 900, 840, 800 cm⁻¹ h) ¹H NMR absorption spectrum : (D₂O) (as shown in Figure 1) δppm : 212 (6H, s), 3.51 (3H, s), 3.72-3.76 (1H, m), 3.99-4.15 (4H, m), 4.18-4.39 (3H, m), 4.62-4.65 (1H, m), 5.62 (1H, d, J=4.3Hz), 6.86-6.95 (2H, m), 7.20-7.25 (2H, m) i) Solubility : Soluble : Water, methanol Insoluble : Acetone, ethyl acetate, chloroform j) Color reaction : Positive : Ferric chloride reaction, reaction with cerium sulfate and iodine vapor Negative : Molish's reaction, Ninhydrin reaction k) Property of substance : Acidic substance From the above physicochemical properties and other investigations, it can be judged that the WS-1627A is a known antibiotic, KA-3093 [Cf. Japan Kokai No. 99495/1981]. WS-1627B a) Appearance : White powder b) Melting point : 107-110°C c) Optical rotation : [α] 20 / D = -126.5° (C=1.0, H₂O) d) Molecular weight : 511 [SIMS : m/z 512 (M⁺ + 1)] e) Elemental analysis (%) : C 53.83; H 5.86; N 2.63 f) UV absorption spectrum : 300 (sh, 22700), 270 (34700), 214 (46000) 300 (sh, 22500), 270 (34500), 214 (45800) 323 (15300), 285 (sh, 25800), 249 (45200), 232 (sh, 41400) g) IR absorption spectrum : 3700-3000, 2920, 1720, 1700, 1640, 1610, 1510, 1430, 1360, 1320, 1270, 1250, 1210, 1160, 1130, 1100, 1060, 1000, 960, 920, 840, 810 cm⁻¹ h) ¹H NMR absorption spectrum : (D₂O) δppm : 2.12 (6H, s), 3.80-3.84 (1H, m), 3.95-4.00 (1H, m), 4.17-4.40 (6H, m), 4.49-4.53 (1H, m), 4.80 (1H, s), 5.23 (1H, s), 5.62 (1H, d, J=4 .3Hz), 6.87-6.95 (2H, m), 7.20-7.26 (2H, m) i) Solubility : Soluble : Water, methanol Insoluble : Acetone, ethyl acetate, chloroform j) Color reaction : Positive : Ferric chloride reaction, reaction with cerium sulfate and iodine vapor Negative : Molish's reaction, Ninhydrin reaction k) Property of substance : Acidic substance From the above physicochemical properties and other investigations, it can be judged that the WS-1627B is a known antibiotic, hygromycin [cf. THE MERCK INDEX TENTH EDITION Page 708 (1983)]. WS-1627C a) Appearance : White powder b) Melting point : 110-113°C c) Optical rotation : [α] 20 / D = -62.0° (C=1.0, H₂O) d) Molecular weight : 513 [SIMS : m/z 514 (M⁺ + 1)] e) Elemental analysis (%) : C 53.65; H 6.33; N 2.95 f) UV absorption spectrum : 300 (sh, 22,750), 270 (34,750), 214 (46,000) 300 (sh, 22,500), 270 (34,750), 214 (46,000) 323 (15,350), 285 (sh, 25,800), 249 (45,200), 232 (sh, 41,400) g) IR absorption spectrum : 3700-3000, 2900, 2700, 1720, 1700, 1640, 1600, 1510, 1420, 1350, 1330, 1260, 1250, 1200, 1120, 1100, 1050, 1020, 960, 900, 830, 800 cm⁻¹ h) ¹H NMR absorption spectrum : (CD₃OD) (as shown in Figure 2) δppm : 2.12 (3H, s), 2.21 (3H, s), 3.51 (3H, s), 3.73-3.75 (1H, m), 4.00-4.14 (4H, m), 4.20-4.30 (2H, m), 4.55-4.65 (2H, m), 5.83 (1H, d, J=4Hz), 6.85-6.94 (2H, m), 7.21-7.25 (2H, m) i) Solubility : Soluble : Water, methanol Insoluble : Acetone, ethyl acetate, chloroform j) Color reaction : Positive : Ferric chloride reaction, reaction with cerium sulfate and iodine vapor Negative : Molish's reaction,Ninhydrin reaction k) Property of substance : Acidic substance From the above physicochemical properties and other investigations, it can be judged that the WS-1627 C is a new epimer of KA-3093. WS-1627D a) Appearance : White powder b) Melting point : 114-117°C c) Optical rotation : [α] 20 / D = -93.1° (C=1.0, H₂O) d) Molecular weight : 511 [m/z 512 (M⁺ + 1)] e) Elemental analysis (%) : C 53.91; H 5.82; N 2.80 f) UV absorption spectrum : 300 (sh, 22,700), 270 (34,700), 214 (46,000) 300 (sh, 22,500), 270 (34,600), 214 (45,900) 323 (sh, 15,300), 285 (sh, 25,900), 249 (45,400), 232 (41,400) g) IR absorption spectrum : 3700-3000, 2900, 1720, 1710, 1640, 1620, 1600, 1510, 1430, 1350, 1270, 1250, 1210, 1130, 1100, 1050, 1020, 960, 920, 900, 880, 840, 810 cm⁻¹ h) ¹H NMR absorption spectrum : (CD₃OD) δppm : 2.12 (3H, m), 2.21 (3H, s), 3.80-3.82 (1H, m), 3.94-4.00 (1H, m), 4.16-4.29 (4H, m), 4.49-4.59 (2H, m), 4.79 (1H, s), 5.23 (1H, s), 5.82 (1H, d, J=4Hz), 6.85-6.89 (2H, m), 7.20-7.25 (2H, m) i) Solubility : Soluble : Water, methanol Insoluble : Acetone, ethyl acetate, chloroform j) Color reaction : Positive : Ferric chloride reaction, reaction with cerium sulfate and iodine vapor Negative : Molish's reaction, Ninhydrin reaction k) Properties of substance : Acidic substance From the above physicochemical properties and other investigations,it can be judged that WS-1627D is a known antibiotic, epi-hygromycin [cf. THE JOURNAL OF ANTIBIOTICS VOL. XXXIII Pages 695 (1980)]. From the above data, chemical structures of WS-1627A, WS-1627B, WS-1627C and WS-1627D can be represented as follows. The WS-1627A, WS-1627B, WS-1627C and WS-1627D can be prepared by culturing a WS-1627A, WS-1627B, WS-1627C and/or WS-1627D-producing strain belonging to the genus Streptomyces in a nutrient medium and recovering the WS-1627A, WS-1627B, WS-1627C and WS-1627D from the cultured broth. Among a WS-1627A, WS-1627B, WS-1627C and/or WS-1627D-producing strain belonging to the genus Streptomyces , Streptomyces noboritoensis subsp. amamiensis No. 1627 was newly isolated from a soil sample collected at Amami-Oshima, Japan. Lyophilized samples of the newly isolated Streptomyces noboritoensis subsp. amamiensis No. 1627 have been deposited with an International Depositary Authority on Budapest Treaty, Fermentation Research Institute, Agency of Industrial Science and Technology residing at Yatabe-cho higashi No. 1-1-3, Tsukuba-gun, Ibaraki-ken, Japan, under the number FERM BP-1275 (formerly, FERM P-8069) since January 25, 1985). Further, Streptomyces hygroscopicus IFO 13472 can also produce the WS-1627A, WS-1627B, WS-1627C and WS-1627D in the cultured broth thereof. It is to be understood that the production of the WS-1627A, WS-1627B, WS-1627C and WS-1627D is not limited to the use of the particular organisms described herein, which are given for illustrative purpose only. Further, it would be possible to make use of any mutants which can be produced from the described organisms by conventional means, such as X-rays, ultra-violet radiation, treatment with N-methyl-N'-nitro-N-nitrosoguanidine, 2-aminopurine and the like. Streptomyces noboritoensis subsp. amamiensis No. 1627 has the following morphological, cultural and physiological characteristics. Morphological observations were made with light and electron microscopes from cultures grown at 30°C for 14 days on yeast-malt extract agar, oatmeal agar and inorganic salts-starch agar. The mature sporophores were formed Rectiflexibiles with more than 30 spores in each chain. Aerial mycelium was poorly developed on some of examined media. The spore surfaces were smooth. Cultural characteristics were observed on ten kinds of media described by Shirling and Gottlieb [Vide. International Journal of Systematic Bacteriology 16, 313-340 (1966)] and by Waksman [Vide. S.A. Waksman "The Actinomycetes Vol. 2 (1961)]. The incubation was made at 30°C for 14 days. The color names used in this study were based on Color Standard (Nihon Shikisai Co., Ltd.). Colonies belonged to the gray or red color series when grown on yeast-malt extract agar and inorganic salt-starch agar. Melanoid pigment was produced on tyrosine agar, but not on peptone-yeast extract-iron agar. Soluble pigment was produced in some of examined media. The results are shown in the following Table 1 in comparison with characteristics of Streptomyces noboritoensis IFO 13065. Cell wall analysis was performed by the methods of Becker et al [Vide. Applied Microbiology 12 , 421-423 (1964)] and Yamaguchi [Vide. Journal of Bacteriology 89 , 444-453 (1965)]. Analysis of whole cell hydrolysates showed the presence of LL-diaminopimelic acid. Accordingly, the cell wall of this strain is believed to be of type I. Physiological properties of the strain No. 1627 were determined according to Shirling and Gottlieb [Vide. the same literature as mentioned above]. Temperature range for growth was from 12°C to 36°C with optimum temperature from 26°C to 28°C. Starch hydrolysis, melanin production and gelatin liquefaction were positive. The results are summarized in Table 2 in comparison with characteristics of Streptomyces noboritoensis IFO 13065. Physiological properties of the strain No. 1627 and Streptomyces noboritoensis IFO 13065. No. 1627 IFO 13065 Temperature range for growth 12°C - 36°C 8°C - 38°C Optimum temperature 26°C - 28°C 24°C - 28°C Nitrate reduction Negative Negative Starch hydrolysis Positive Positive Milk coagulation Negative Negative Milk peptonization Negative Positive Melanin production Positive Positive Gelatin liquefaction Positive Positive H₂S production Negative Positive NaCl tolerance 7% <-<10% 7% <-<10% Utilization of carbon sources was examined according to the method of Pridham and Gottlieb [Vide. Journal of Bacteriology 56 , 107-114 (1948)]. The growth was observed after 14 days incubation at 30°C. Carbon sources utilization of this strain is shown in Table 3 in comparison with those of Streptomyces noboritoensis IFO 13065. Carbon sources utilization of strain No. 1627 and Streptomyces noboritoensis IFO 13065. Carbon source No. 1627 IFO 13065 D-Glucose + + Sucrose + + Glycerin + + D-Xylose + + D-fructose - + Lactose + + Maltose + + Rhamnose - - Raffinose + + D-Galactose + + L-Arabinose + + D-Mannose + + D-Trehalose + + Inositol + - Mannitol - + Inulin - - Cellulose - - Salicin - + Chitin - - Sodium citrate + + Sodium succinate + + Sodium acetate ± - Symbols : + = utilization, ± = doubtful utilization, - = no utilization Microscopic studies and cell wall composition analysis indicate that the strain No. 1627 belongs to the genus Streptomyces Waksman and Henrici 1943. Accordingly, a comparison of this strain was made with various Streptomyces species in the light of the published descriptions [Vide. International Journal of Systematic Bacteriology 18 , 69-189, 279-392 (1968) and 19 , 391-512 (1969), and Bergey's Manual of Determinative Bacteriology 8th Edition (1974)]. As the results of the comparison, the strain No. 1627 was considered to resemble Streptomyces noboritoensis Isono et al. 1957. Therefore, the strain No. 1627 was compared with the corresponding S . noboritoensis IFO 13065. As the results of comparison, cultural characteristics of the strain No. 1627 are different from those of S . noboritoensis on inorganic salts-starch agar, potato-dextose agar and peptone-yeast extract-iron agar. Milk peptonization and H₂S production of the strain No. 1627 are negative, but those of S . noboritoensis are positive. In carbon utilization, the strain No. 1627 can utilize inositol, but S . noboritoensis can not utilize it. And, the strain No. 1627 can not utilize D-fructose, mannitol and salicin, but S . noboritoensis can utilize them. As the results of comparison as mentioned above, there are some differences between the strain No. 1627 and S . noboritoensis . However, morphological and cultural characteristics were similar each other on numerous media. Therefore, the strain No. 1627 can be considered to belong to S . noboritoensis . Considering several differences of the strain No. 1627 from S . noboritoensis , it is reasonable that the strain No. 1627 is regarded as new subspecies of S . noboritoensis . Therefore, the strain No. 1627 has been designated as Streptomyces noboritoensis subsp. amamiensis No. 1627. The WS-1627A, B, C and D can be produced by culturing a WS-1627 A, B, C and/or D producing strain belonging to the genus Streptomyces in a nutrient medium. In general, the WS-1627A, B, C and D can be produced by culturing the WS-1627A, B, C and/or D producing strain in a nutrient medium containing assimilable sources of carbon and of nitrogen, preferably under aerobic conditions (e.g. shaking culture, submerged culture, etc.). The preferred sources of carbon in the nutrient medium are carbohydrates such as glucose, fructose, glycerin and starch. Other sources which may be included are lactose, arabinose, xylose, dextrin, molasses and the like. The preferred sources of nitrogen are yeast extract, peptone, gluten meal, cottonseed meal, soybean meal, corn steep liquor, dried yeast, etc., as well as inorganic and organic nitrogen compounds such as ammonium salts (e.g. ammonium nitrate, ammonium sulphate, ammonium phosphate, etc.), urea, amino acid and the like. The carbon and nitrogen sources, though advantageously employed in combination, need not be used in their pure form because less pure materials which contain traces of growth factors and considerable quantities of mineral nitrients, are also suitable for use. When desired, there may be added to medium such mineral salts as calcium carbonate, sodium or potassium phosphate, sodium or potassium iodide, magnesium salt, cobalt chloride and the like. If necessary, especially when the culture medium is foamed remarkably, a defoaming agent such as liquid paraffin, higher alcohol, plant oil, mineral oil and silicones may be added. As conditions for the production in massive amounts, submerged aerobic cultural condition is preferred for the production of the WS-1627A, B, C and D. For the production in small amounts, a shaking or surface culture in a flask or bottle is employed. Furthermore, when the growth is carried out in large tanks, it is preferable to use the vegetative form of the organism for inoculation in the production tanks in order to avoid growth lag in the process of production of the object compounds. Accordingly, it is desirable first to produce a vegetative inoculum of the organism by inoculating a relatively small quantity of culture medium with spores or mycelia of the organism and culturing said inoculated medium, and then to transfer the cultured vegetative inoculum aseptically to large tanks. As the medium, in which the vegetative inoculum is produced, there can be used the substantially same as or somewhat different medium from medium utilized for main production of the object compounds. Agitation and aeration of the culture mixture may be accomplished in a variety of ways. Agitation may be provided by a propeller or the similar mechanical agitation equipment, by revolving or shaking the fermenter, by various pumping equipment or by the passage of sterile air through the medium. Aeration may be effected by passing sterile air through the fermentation mixture. The fermentation is usually conducted at a temperature about between 20°C and 40°C, preferably around 30°C, for a period of 50 hours to 100 hours, which may be varied according to the fermentation conditions and scale. Thus produced WS-1627A, B, C and D can be recovered from the culture medium by conventional means which are commonly used for the recovery of other fermentation products such as antibiotics. In general, most of the WS-1627A, B, C and D produced are found in the culture filtrate, and accordingly the object compounds can be isolated from the filtrate, which is obtained by filtering or centrifuging the broth, by a conventional method such as concentration under reduced pressure, lyophilization, extraction with a conventional solvent, pH adjustment, treatment with a conventional resin (e.g. anion or cation exchange resin, non-ionic adsorption resin), treatment with a conventional adsorbent (e.g. activated charcoal, silicic acid, silica gel, cellulose, alumina), crystallization, recrystallization and the like. The WS-1627A, B, C and D can be separated from each other and isolated, for example, by subjecting a crude material comprising WS-1627A, B, C and D to a low pressure and high performance liquid chromatography using reverse phase silica gel [e.g. Lichroprep RP-8, RP-18 (trademark, made by E. Merck), µBondapak C₁₈ (trademark, made by Waters Associates)]. The WS-1627A, B, C and D as obtained in their free forms may be converted to a salt with an inorganic or organic base such as sodium salt, potassium salt, triethylamine salt and the like. The WS-1627A, B, C and D or their salts can be administered orally or parenterally as a growth promoting agent to growing animals (e.g. piglets, chicks, etc.) in a conventional manner. In cases where the WS-1627A, B, C and D and their salts are orally administered to animals, one or more effective ingredient(s) selected from the WS-1627A, B, C, D and their salts may usually be administered as it is or in admixture with a suitable carrier (e.g. water, kaolin, talc, calcium carbonate, lactose, etc.) or in admixture with a feed. More particularly, the effective ingredient(s) may be administered to animals as a drinking water in the form of aqueous solution; or as a tablet, granule or capsule in the form of preparations which comprise the effective ingredient(s) and suitable non-toxic carrier(s) as exemplified above; or as a ration in the form of the composition which comprises the effective ingredient(s) and animal feed. Further, in cases where the WS-1627A, B, C or D or mixture thereof is parenterally administered to animals, a conventional and suitable injection preparation which comprises the effective ingredient(s) and suitable non-toxic carrier(s) (e.g. water, etc.) can be employed. With regard to the form of administering the feed composition as mentioned above, the ration comprising one or more effective ingredient(s) selected from the WS-1627A, B, C and D and their salts can be prepared in a conventional manner, namely by admixing the effective ingredient(s) with basal ration. The dosage of the effective ingredient(s) to animals may be varied over a very wide range depending upon various factors such as the kind of animal, growth period, administration route and the like. Levels of the effective ingredient(s) in the feed and drinking water which are construed as preferable and anticipated to promote growth of growing animals may be in an amount between about 0.5 and about 250 ppm. Further, in the acute toxicity test, each of five ddy mice (5-week old, male, 20 g in weight), was intravenously given a single dose of WS-1627A, B, C or D 20 mg (lg/kg) and all survived. During 10 days of observation after injection, no toxic symptom was observed. The following Examples are given to illustrate this invention, but it should be understood that they are not intended to limit this invention. Example 1 [Preparation of WS-1627 A and C] An aqueous seed medium (80 ml) containing 1% corn starch, 1% glycerin, 0.5% glucose, 1% cotton seed meal, 0.5% dried yeast, 0.5% corn steep liquor, 0.2% calcium carbonate (pH 6.5) was placed into each of ten 250 ml Erlenmeyer flasks and sterilized at 120°C for 30 minutes. A loopful of Streptomyces noboritoensis subsp. amamiensis No. 1627 on matured slant culture was inoculated to the seed medium. The flasks were shaken on a rotary shaker at 30°C for 3 days. An aqueous production medium (18 ℓ) containing 3% starch, 2% sucrose, 0.5% cotton seed meal, 0.5% dried yeast, 0.5% gluten meal, 0.05% magnesium sulfate heptahydrate, 0.05% sodium chloride, 0.0004% cobaltous chloride hexahydrate, 0.00005% sodium iodide, 0.2% calcium carbonate (pH 6.8) was poured into each of two 30 ℓ stainless steel fermenters and sterilized at 120°C for 30 minutes. The resultant seed culture broth (360 ml) was inoculated to each of the production medium and cultured at 30°C for 3 days, agitated at 300 rpm and aerated at 20 liters per minute. The cultured broth (35 ℓ) was filtrated with the aid of diatomaceous earth (500 g). The filtrate (32 ℓ, pH 7.2) was passed through a column of activated charcoal (3.2 ℓ). The column was washed with water (7 ℓ) and eluted with 60% acueous acetone (15 ℓ). The eluate was concentrated to a volume of 1 liter under reduced pressure. The concentrate was chromatographed on CM-Sephadex C-25 (H⁺, made by Pharmacia Fine Chemicals A B) column (2 ℓ). The column was developed with water. The active fraction (4 ℓ) was concentrated under reduced pressure. The residue was chromatographed on a column (200 ml) of silica gel using saturated aqueous n-butanol as developing solvent. The active fraction (320 ml) was combined and concentrated to a volume of 50 ml under reduced pressure. The resultant solution was chromatographed on a column (50 ml) of nonionic adsorption resin, HP-20 (trademark, made by Mitsubishi Chemical Industries Ltd.) to remove the silica gel. The column was washed with water (200 ml) and 20% aqueous methanol (200 ml) and then eluted with 40% aqueous methanol (250 ml). The eluate was concentrated under reduced pressure and the resultant solution was freeze-dried to give a pale yellow powder (900 mg). The crude powder was dissolved in 30% aqueous methanol (9 ml) and applied to a column of low pressure liquid chromatography using reverse phase silica gel [Lichroprep RP-8 (trademark) made by E. Merck, size B : 310 x 25 mm, 40-63 µm, detected by UV absorbance at 254 nm, flow rate 10 ml/min) with 30% aqueous methanol. The first fractions (from 220 ml to 290 ml) were collected and concentrated under reduced pressure. The resultant solution was freeze-dried to give a white powder (Powder I, 150 mg) containing WS-1627A and WS-1627C. The continuous fractions (from 310 ml to 360 ml) were collected and concentrated under reduced pressure. The resultant solution was freeze-dried to give a white powder (Powder II, 12.7 mg) containing WS-1627B and WS-1627D. The powder I (140 mg) was further purified, in seven separate runs, using a high performance liquid chromatography. Each portion (20 mg) of the starting charge was dissolved in 20% aqueous methanol (0.2 ml) and applied to a column of reverse phase silica gel (µBondapak C₁₈ (trademark) made by Waters Associates, 7.8 x 30 mm, 8-10 µm, detected by UV absorbance at 254 nm, flow rate 2.5 ml/min) with 20% aqueous methanol. The first fractions (from 45 ml to 50 ml) of each of the seven chromatographic separation were combined and concentrated under reduced pressure. The resultant solution was freeze-dried to give a white powder (15.8 mg) of WS-1627C. The continuous fractions (from 52 ml to 60 ml) of each of the seven chromatographic separations were combined and concentrated under reduced pressure. The resultant solution was freeze-dried to give a white powder (117 mg) of WS-1627A. Example 2 [Preparation of WS-1627B and D] An aqueous seed medium (160 ml) containing 1% corn starch, 1% glycerin, 0.5% glucose, 1% cotton seed meal, 0.5% dried yeast, 0.5% corn steep liquor, 0.2% calcium carbonate (pH 6.5) into each of twenty three 500 ml Erlenmeyer flasks and sterilized at 120°C for 30 minutes. A loopful of Streptomyces noboritoensis subsp. amamiensis No. 1627 on matured slant culture was inoculated to the seed medium. The flasks were shaken on a rotary shaker at 30°C for 3 days. An aqueous production medium (180 ℓ) containing 3% starch, 2% glycerin, 0.5% soy bean meal, 2% gluten meal, 0.05% magnesium sulfate heptahydrate, 0.05% sodium chloride, 0.0004% cobaltous chloride hexahydrate, 0.00005% sodium iodide, 0.2% calcium carbonate (pH 6.8) was poured into a 200 ℓ stainless steel fermenter and sterilized at 120°C for minutes. The resultant seed culture broth (3.6 ℓ) was inoculated to the production medium and cultured at 30°C for 4 days, agitated at 250 rpm and aerated at 20 liters per minute. The culture broth (180 ℓ) was filtrated with the aid of diatomaceous earth (2.5 kg). The filtrate (170 ℓ,pH 7.2) was passed through a column (15 ℓ) of activated charcoal. The column was washed with water (60 ℓ) and 20% aqueous acetone (60 ℓ) and then eluted with 60% aqueous acetone (75 ℓ). The eluate was concentrated to a volume of 17 liters under reduced pressure. The concentrate was chromatographed on a column (4 ℓ) of CM-Sephadex C-25 (H⁺). The column was developed with water. The activated fractions (3 ℓ) was combined and concentrated under reduced pressure. The residue was chromatographed on a column (200 ml) of silica gel using saturated aqueous n-butanol as developing solvent. The active fractions were combined and concentrated to a volume of 100 ml under reduced pressure. The resultant solution was chromatographed on a column of HP-20 (200 ml) to remove the silica gel. The column was washed with water (800 ml) and 20% aqueous methanol (1200 ml) and then eluted with 40% aqueous methanol (1200 ml). The eluate was freeze-dried to give a pale yellow powder (3.62 g). The crude powder was dissolved in 30% aqueous methanol (36 ml) and applied to a column of low pressure liquid chromatography using reverse phase silica gel [Lichroprep RP-8 (trademark) made by E. Merck, size B : 310 x 25 mm, 40-63 µm, detected by UV absorbance at 254 nm, flow rate 10 ml/min] with 30% aqueous methanol, in four separate runs. The first fractions (from 250 ml to 275 ml) of each of the four chromatographic separations were combined and concentrated under reduced pressure. The resultant solution was freeze-dried to give a white powder (Powder I, 108 mg) containing WS-1627A and WS-1627C. The continuous fractions (from 285 ml to 370 ml) of each of the four chromatographic separations were combined and concentrated under reduced pressure. The resultant solution was freeze-dried to give a white powder (Powder II, 2.07 g) containing WS-1627B and WS-1627D. The powder II (500 mg) was further purificated, in twenty five separated runs, using a high performance liquid chromatography. Each portion (20 mg) of the starting charge was dissolved in 20% aqueous methanol (0.2 ml) and applied to a column of reverse phase silica gel (µBondapak C₁₈ (trademark) made by Waters Associates, 7.8 x 30 mm, 8 x 10 µm, detected by UV absorbance at 254 nm, flow rate 2.5 ml/min) with 20% aqueous methanol. The first fractions (from 83 ml to 88 ml) of each of the twenty five chromatographic separations were combined and concentrated under reduced pressure. The resultant solution was freeze-dried to give a white powder (350 mg) of WS-1627D. The continuous fractions (from 93 ml to 100 ml) of each of twenty five chromatographic separations were combined and concentrated under reduced pressure. The resultant solution was freeze-dried to give a white powder (407 mg) of WS-1627B. Example 3 [Preparation of WS-1627A, B, C and D] The WS-1627A, B, C and D were prepared in substantially the same manner as those of Example 1 and 2 except using Streptomyces hygroscopicus IFO 13472 in place of Streptomyces noboritoensis subsp. amamiensis No. 1627. Example 4 [Growth promoting effect] Five sets of 24 pigs (33 days-old, LWD strain) were used. Each 24 pigs were divided into 4 groups, i.e. 3 treatment groups and a control group. Each group consisted of 3 barrows and 3 female pigs. They were fed continuously with the feed described in Table 4. Their body weight and feed consumption were observed for 6 weeks. The results are shown in Table 5. Basal ration I Ingredient % Yellow maize 63 Wheat flour 8 Defatted Soybean meal 16 Skim milk 5 Fish meal 3 Vegetable oil 1.5 Tricalcium phosphate 0.8 Calcium carbonate 1.2 Sodium chloride 0.4 Zeolite 1 Nutritious premix 0.1 Feed composition fed to animals Feed Composition fed to Control group The above basal ration I Feed Composition fed to Treatment group I The above basal ration I + WS-1627B 10 ppm Feed Composition fed to Treatment group II The above basal ration I + WS-1627 B 20 ppm Feed Composition fed to Treatment group III The above basal ration I + WS-1627 B 40 ppm Example 5 [Growth promoting effect] 8-Days-old male broiler chickens (chunky) were divided into 3 groups i.e. 2 treatment groups and a control group, each group of which consisted of 10 chicks. They were fed continuously with the feed described in Table 6. Their body weight and feed consumption were observed for one week. The results are shown in Table 7. Basal ration II Ingredient % Sucrose 55.7 Soybean oil meal 29.3 Alfalfa meal 2.0 white fish meal 8.0 Animal fat 3.2 Calcium carbonate 0.3 Calcium phosphate 0.7 Salt 0.2 DL-methionine 0.15 Choline hydrochloride 0.05 Vitamin premix 0.2 Mineral premix 0.2 Feed composition fed to animals Feed composition fed to Control group The above Basal ration II Feed composition fed to Treatment group I The above Basal ration II + WS-1627 B 10 ppm Feed composition fed to Treatment group II The above Basal ration II + WS-1627 B 50 ppm Example 6 [Injection Preparation] Some sterile samples of WS-1627B are distributed into vials, thereby containing 100 mg of the active ingredient. The vials are sealed hermetically to exclude bacteria. Whenever the vials are required for use, 2 ml of sterile distilled water for injection is added to the vial and the vial is subjected to administration. Example 7 [Feed preparation] Ingredient Skim milk 270 kg Dried whey 100 kg fish meal 50 kg Wheat flour 350 kg Glucose 50 kg Tallow 30 kg Dried yeast 100 kg Starch 21 kg Casein sodium 10 kg Tricalcium phosphate 10 kg Sodium chloride 4 kg DL-Methionine 500 g Lysine hydrochloride 1 kg Vitamin A D E premix 2 kg Vitamin B premix 2 kg Trace mineral premix 1 kg WS-1627 B 100 g

Claims: Claims for the following Contracting States : AT, BE, CH, DE, FR, GB, IT, LI, LU, NL, SE Use of one or more compound(s) selected from WS-1627A, WS-1627B, WS-1627C and WS-1627D, or their salts, for the manufacture of a growth promoting agent for the treatment of animals. Use as claimed in claim 1, wherein the growth promoting agent also comprises one or more non-toxic carriers. Use as claimed in claim 1 or claim 2, wherein the growth promoting agent is formulated for oral administration. Use as claimed in claim 3, wherein the growth promoting agent is formulated as an aqueous solution. Use as claimed in claim 3, wherein the growth promoting agent is formulated for admixture with animal feed. Use as claimed in claim 1 or claim 2, wherein the growth promoting agent is formulated for parenteral administration. A foodstuff for animals comprising one or more compounds selected from WS-1627A, WS-1627B, WS-1627C and WS-1627D, or their salts. Claims for the following Contracting States : ES, GR A process for the manufacture of a growth promoting agent for the treatment of animals which comprises the use of one or more compounds selected from WS-1627A, WS-1627B, WS-1627C and WS-1627D, or their salts. A process as claimed in claim 1, wherein the growth promoting agent also comprises one or more non-toxic carriers. A process as claimed in claim 1 or claim 2, wherein the growth promoting agent is formulated for oral administration. A process as claimed in claim 3, wherein the growth promoting agent is formulated as an aqueous solution. A process as claimed in claim 3, wherein the growth promoting agent is formulated for admixture with animal feed. A process as claimed in claim 1 or claim 2, wherein the growth promoting agent is formulated for parenteral administration.

Inventors: HASHIMOTO, SEIJI, KOHSAKA, MASANOBU, KUNUGITA, KIYOHIKO, UMEHARA, KAZUYOSHI, YAMASHITA, MICHIO
Applicants: FUJISAWA PHARMACEUTICAL CO., LTD.
Application year: 1987
Main classification: A61K31
IPC codes: A61P3, C12P1, A61P31, C12N1, A61K35, C07G11, C07H15, C12P19, C12R1