JPH0445516B2 - - Google Patents
Info
- Publication number
- JPH0445516B2 JPH0445516B2 JP58234037A JP23403783A JPH0445516B2 JP H0445516 B2 JPH0445516 B2 JP H0445516B2 JP 58234037 A JP58234037 A JP 58234037A JP 23403783 A JP23403783 A JP 23403783A JP H0445516 B2 JPH0445516 B2 JP H0445516B2
- Authority
- JP
- Japan
- Prior art keywords
- spirocardin
- culture
- spirocardine
- eluted
- medium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
- BHDYBYAEPPUJEJ-UHFFFAOYSA-N 4-[(4,5-dihydroxy-1,6-dioxaspiro[2.4]heptan-7-yl)methyl]-2-hydroxy-3,4,8,8a-tetramethyl-3,4a,5,6-tetrahydro-2h-naphthalen-1-one Chemical compound CC1C(O)C(=O)C(C(=CCC2)C)(C)C2C1(C)CC1OC(O)C(O)C11CO1 BHDYBYAEPPUJEJ-UHFFFAOYSA-N 0.000 claims description 54
- PDPQILPMCDOHDB-UHFFFAOYSA-N 4-[2-[2-(1,2-dihydroxyethyl)oxiran-2-yl]-2-hydroxyethyl]-2-hydroxy-3,4,8,8a-tetramethyl-3,4a,5,6-tetrahydro-2h-naphthalen-1-one Chemical compound CC1C(O)C(=O)C(C(=CCC2)C)(C)C2C1(C)CC(O)C1(C(O)CO)CO1 PDPQILPMCDOHDB-UHFFFAOYSA-N 0.000 claims description 24
- 241000894006 Bacteria Species 0.000 claims description 9
- 241000187654 Nocardia Species 0.000 claims description 7
- 241000187681 Nocardia sp. Species 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 4
- 238000012258 culturing Methods 0.000 claims 1
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 39
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 33
- 239000002609 medium Substances 0.000 description 26
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 24
- 239000000243 solution Substances 0.000 description 16
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 14
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 12
- 238000000862 absorption spectrum Methods 0.000 description 12
- 239000000741 silica gel Substances 0.000 description 11
- 229910002027 silica gel Inorganic materials 0.000 description 11
- 239000000126 substance Substances 0.000 description 11
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 10
- 229920001817 Agar Polymers 0.000 description 8
- 239000008272 agar Substances 0.000 description 8
- 238000000034 method Methods 0.000 description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 7
- 210000004027 cell Anatomy 0.000 description 7
- 239000007788 liquid Substances 0.000 description 7
- 239000003921 oil Substances 0.000 description 7
- 235000019198 oils Nutrition 0.000 description 7
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 6
- 239000000284 extract Substances 0.000 description 6
- 239000012046 mixed solvent Substances 0.000 description 6
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 6
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 6
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 5
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 5
- 239000003463 adsorbent Substances 0.000 description 5
- 238000005273 aeration Methods 0.000 description 5
- 230000001580 bacterial effect Effects 0.000 description 5
- 229940041514 candida albicans extract Drugs 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 239000012138 yeast extract Substances 0.000 description 5
- 241000186361 Actinobacteria <class> Species 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 4
- 235000013312 flour Nutrition 0.000 description 4
- 230000007935 neutral effect Effects 0.000 description 4
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- 235000002639 sodium chloride Nutrition 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- CZDYPVPMEAXLPK-UHFFFAOYSA-N tetramethylsilane Chemical compound C[Si](C)(C)C CZDYPVPMEAXLPK-UHFFFAOYSA-N 0.000 description 4
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- 239000003242 anti bacterial agent Substances 0.000 description 3
- 230000000844 anti-bacterial effect Effects 0.000 description 3
- 239000002518 antifoaming agent Substances 0.000 description 3
- 230000003115 biocidal effect Effects 0.000 description 3
- 229910000019 calcium carbonate Inorganic materials 0.000 description 3
- 210000002421 cell wall Anatomy 0.000 description 3
- 238000004440 column chromatography Methods 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000003960 organic solvent Substances 0.000 description 3
- 238000001179 sorption measurement Methods 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241001061260 Emmelichthys struhsakeri Species 0.000 description 2
- 244000068988 Glycine max Species 0.000 description 2
- 235000010469 Glycine max Nutrition 0.000 description 2
- 241000204031 Mycoplasma Species 0.000 description 2
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
- 229920006705 PC-G Polymers 0.000 description 2
- 239000006159 Sabouraud's agar Substances 0.000 description 2
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 2
- 241000187747 Streptomyces Species 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical compound [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 238000013019 agitation Methods 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- OSASVXMJTNOKOY-UHFFFAOYSA-N chlorobutanol Chemical compound CC(C)(O)C(Cl)(Cl)Cl OSASVXMJTNOKOY-UHFFFAOYSA-N 0.000 description 2
- 235000012343 cottonseed oil Nutrition 0.000 description 2
- 238000003113 dilution method Methods 0.000 description 2
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 2
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 2
- 235000019797 dipotassium phosphate Nutrition 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 235000011187 glycerol Nutrition 0.000 description 2
- 238000004896 high resolution mass spectrometry Methods 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 150000002632 lipids Chemical class 0.000 description 2
- 235000013372 meat Nutrition 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 230000000877 morphologic effect Effects 0.000 description 2
- 230000001766 physiological effect Effects 0.000 description 2
- 229910052697 platinum Inorganic materials 0.000 description 2
- 239000012286 potassium permanganate Substances 0.000 description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000012449 sabouraud dextrose agar Substances 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 description 2
- HPALAKNZSZLMCH-UHFFFAOYSA-M sodium;chloride;hydrate Chemical class O.[Na+].[Cl-] HPALAKNZSZLMCH-UHFFFAOYSA-M 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 238000004809 thin layer chromatography Methods 0.000 description 2
- 241001148471 unidentified anaerobic bacterium Species 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- GMKMEZVLHJARHF-UHFFFAOYSA-N (2R,6R)-form-2.6-Diaminoheptanedioic acid Natural products OC(=O)C(N)CCCC(N)C(O)=O GMKMEZVLHJARHF-UHFFFAOYSA-N 0.000 description 1
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 description 1
- 101100313763 Arabidopsis thaliana TIM22-2 gene Proteins 0.000 description 1
- 235000017060 Arachis glabrata Nutrition 0.000 description 1
- 244000105624 Arachis hypogaea Species 0.000 description 1
- 235000010777 Arachis hypogaea Nutrition 0.000 description 1
- 235000018262 Arachis monticola Nutrition 0.000 description 1
- 229920001353 Dextrin Polymers 0.000 description 1
- 239000004375 Dextrin Substances 0.000 description 1
- 241001453233 Doodia media Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 235000019733 Fish meal Nutrition 0.000 description 1
- 241000282414 Homo sapiens Species 0.000 description 1
- 239000005909 Kieselgur Substances 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241001655308 Nocardiaceae Species 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 240000008042 Zea mays Species 0.000 description 1
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 1
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 1
- -1 acid preparations Substances 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 1
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 description 1
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 1
- KTUQUZJOVNIKNZ-UHFFFAOYSA-N butan-1-ol;hydrate Chemical compound O.CCCCO KTUQUZJOVNIKNZ-UHFFFAOYSA-N 0.000 description 1
- 235000010216 calcium carbonate Nutrition 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 229920001429 chelating resin Polymers 0.000 description 1
- 229960004926 chlorobutanol Drugs 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 239000000306 component Substances 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 239000002385 cottonseed oil Substances 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 235000019425 dextrin Nutrition 0.000 description 1
- 229940079920 digestives acid preparations Drugs 0.000 description 1
- 239000004467 fishmeal Substances 0.000 description 1
- 229930182830 galactose Natural products 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 239000010439 graphite Substances 0.000 description 1
- 229910002804 graphite Inorganic materials 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 239000012533 medium component Substances 0.000 description 1
- GMKMEZVLHJARHF-SYDPRGILSA-N meso-2,6-diaminopimelic acid Chemical compound [O-]C(=O)[C@@H]([NH3+])CCC[C@@H]([NH3+])C([O-])=O GMKMEZVLHJARHF-SYDPRGILSA-N 0.000 description 1
- XELZGAJCZANUQH-UHFFFAOYSA-N methyl 1-acetylthieno[3,2-c]pyrazole-5-carboxylate Chemical compound CC(=O)N1N=CC2=C1C=C(C(=O)OC)S2 XELZGAJCZANUQH-UHFFFAOYSA-N 0.000 description 1
- 239000010446 mirabilite Substances 0.000 description 1
- 235000013379 molasses Nutrition 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 238000004816 paper chromatography Methods 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 238000005192 partition Methods 0.000 description 1
- 235000020232 peanut Nutrition 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 235000021317 phosphate Nutrition 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- OVARTBFNCCXQKS-UHFFFAOYSA-N propan-2-one;hydrate Chemical compound O.CC(C)=O OVARTBFNCCXQKS-UHFFFAOYSA-N 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 1
- 229920002545 silicone oil Polymers 0.000 description 1
- 235000010344 sodium nitrate Nutrition 0.000 description 1
- 239000004317 sodium nitrate Substances 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- RSIJVJUOQBWMIM-UHFFFAOYSA-L sodium sulfate decahydrate Chemical compound O.O.O.O.O.O.O.O.O.O.[Na+].[Na+].[O-]S([O-])(=O)=O RSIJVJUOQBWMIM-UHFFFAOYSA-L 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 235000012424 soybean oil Nutrition 0.000 description 1
- 239000003549 soybean oil Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- FKHIFSZMMVMEQY-UHFFFAOYSA-N talc Chemical compound [Mg+2].[O-][Si]([O-])=O FKHIFSZMMVMEQY-UHFFFAOYSA-N 0.000 description 1
- 229910021654 trace metal Inorganic materials 0.000 description 1
- 241001446247 uncultured actinomycete Species 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Epoxy Compounds (AREA)
- Saccharide Compounds (AREA)
- Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Compounds Of Unknown Constitution (AREA)
Description
本発明は新抗生物質スピロカルデイン
(Spirocardin)AまたはB並びにその製造法に関
するものである。本発明者らは福島県耶麻郡で採
取した土壌から分離したノカルジア属に属する
SANK64282株がグラム陽性、グラム陰性殺菌に
対して有効な新抗生物質がスピロカルデインAま
たはBを生産することを見出した。
従つて、スピロカルデインAまたはBはこれら
細菌に起因するヒト、動物または植物の疾病の予
防または治療に用いられる。
ここに、スピロカルデインAまたはBは下記の
式で表わされる。
但し、式中、スピロカルデインAはXが基
The present invention relates to a new antibiotic Spirocardin A or B and a method for producing the same. The present inventors isolated a specimen belonging to the genus Nocardia from soil collected in Yama-gun, Fukushima Prefecture.
We found that the SANK64282 strain produces spirocardin A or B, a new antibiotic that is effective against Gram-positive and Gram-negative bactericidal bacteria. Therefore, spirocardin A or B can be used for the prevention or treatment of human, animal or plant diseases caused by these bacteria. Here, spirocardine A or B is represented by the following formula. However, in the formula, spirocardine A is
【式】を示し、スピロカルデインB はXが基[Formula] is shown, spirocardine B is based on X
【式】を示す。
スピロカルデインAまたはBを生産する
SANK64282株の菌学的性状は次の通りである。
(1) 形態学的特徴
ISP〔インターナシヨナル・ストレプトミセ
ス・プロジエクト(International
Streptomyces Project)〕規定の倍地およびワ
ツクスマン(S.A.Waksman)らの勧告〔アク
チノミセイテス(The Actinomycetes)2巻〕
の倍地上で培養する。培養は通常28℃で行う。
SANK64282株の形態学的特徴は、接種培養の
数日後に気菌糸および栄養菌糸に明瞭な分断が
観察されることである。分断菌糸は球状ないし
桿状を示す。その他、特殊器官の形成は観察さ
れない。
(2) 各種培養基上の諸性状
各種培養基上で28℃、14日間培養したときの
性状は第1表に示す通りである。色調の表示は
日本色彩研究所版“標準色票”のカラーチツプ
ナンバーを表わす。[Formula] is shown. Produce spirocardin A or B
The mycological properties of SANK64282 strain are as follows. (1) Morphological characteristics ISP [International Streptomyces projectus]
Streptomyces Project] Prescribed double areas and recommendations of SAWaksman et al. [The Actinomycetes, vol. 2]
Culture on medium. Culture is usually carried out at 28°C.
A morphological feature of the SANK64282 strain is that clear division between aerial and vegetative hyphae is observed several days after inoculation and culture. The segmented hyphae are spherical or rod-shaped. No other special organs were observed. (2) Properties on various culture media Properties when cultured on various culture media at 28°C for 14 days are shown in Table 1. The color tone display represents the color chip number of the Japan Color Research Institute's "Standard Color Chart".
【表】【table】
【表】【table】
【表】
(3) 生理的性質
SANK 64282株の生理学的性質を第2表に、
炭素源資化性を第3表に示す。[Table] (3) Physiological properties The physiological properties of SANK 64282 strain are shown in Table 2.
Table 3 shows the carbon source utilization.
【表】【table】
【表】【table】
【表】
+:同化、±:弱く資化、−:同化
せず
(4) 菌体内成分について
エム・ピー・レシエバリヤー(M.P.
Lechevalier)らの方法〔エイ・デイーツ(A.
Dietz)ら著、放線菌の分類(Actinomycete
taxonomy)、225頁、1980年〕に従い、菌体の
酸加水分解物のペーパークロマトグラフイーに
よる分析を行つた結果、メソジアミノピメリン
酸およびアラビノース、ガラクトースが認めら
れ、細胞壁型のタイプは型であることが確認
された。また全菌体糖型はA型であつた。さら
にエツチ・モルダルスカ(H.Mordarska)ら
の方法〔ジヤーナル・オブ・ジエネラル・マイ
クロバイオロジー(Journal of general
Microbiology)、71巻、77頁、1972年〕に従
い、菌体内の脂質を分析した結果、LCN−A
(Lipids characteristic of Nocardia)が存在
していた。
上記の如きSANK 64282株の諸性状のうち特
に菌糸が分断すること、細胞壁が型で全菌
体中の糖型がA型であること、菌体内にLCN
−Aが存在すること等から、本菌株は、放線菌の
中でもノカルジアセアエ(Nocardiaceae)科の
ノカルジア(Nocardia)属に属する菌株である
ことが明らかとなり、ノカルジア・エスピー
(Nocardia sp.)SANK 64282と命名した。
本菌株は通産省工業技術院微生物工業技術研究
所に寄託されておりその微生物受託番号は第6986
号である。
以上、スピロカルデインAまたはBの生産菌に
ついて説明したが、放線菌、特にノカルジア属の
諸性質は一定したものでなく、自然的、人工的に
容易に変化することは周知の通りであり、本発明
で使用しうる菌はノカルジア属に属するスピロカ
ルデインAまたはBを生産するすべての菌株を包
含するものである。
本発明における培養は、一般放線菌における培
養方法に準じて行われ、液体培地中での振盪培養
あるいは通気撹拌培養によるのが好ましい。培地
成分としては、たとえば炭素源としてブドウ糖、
マルトース、シユクロース、マンニツト、糖蜜、
グリセリン、デキストリン、澱粉、大豆油、綿実
油などが、窒素源として大豆粉、落花生粉、綿実
粉、フアーマミン、魚粉、コーン・スチープ・リ
カー、ペプトン、肉エキス、イースト、イース
ト・エキス、硝酸ソーダ、硝酸アンモニウム、硫
酸アンモニウムなどが、また無機塩として食塩、
燐酸塩、炭酸カルシウム、微量金属塩などが必要
に応じて適宜添加される。液体培養に際しては、
シリコン油、植物油、界面活性剤等が消泡剤とし
て適宜使用される。
培地のPHは中性附近、培養温度は24℃から30
℃、特に28℃前後が好ましい。
培養の経過に伴つて生産されるスピロカルデイ
ンAまたはBの力価の経時的変化は、スタフイロ
コツカス・アウレウス(Staphylococcus
aureus)FDA 209P JC−1を被検菌としてペー
パーデイスク(東洋科学産業(株)製、直径8mm、
Thick)検定法により測定される。
通常72〜120時間の培養でスピロカルデインA
またはBの生産量は最高値に達する。主として培
養液中の液体部分に存在するスピロカルデインA
またはBは、培養終了後、菌体その他の固型部分
をけいそう土等を過助剤とする過操作、ある
いは遠心分離によつて除去し、その液あるいは
上清中から抽出、精製することによつて得られ
る。
スピロカルデインAまたはBは、その物理化学
的性状を利用することにより、たとえば吸着剤を
用いて採取することができる。吸着剤としてはた
とえば活性炭、あるいは吸着用樹脂であるアンバ
ーライトXAD−2、XAD−4、XAD−7等
(ローム・アンド・ハース社製)またはダイヤイ
オンHP10,HP20,HP20AG,HP50等(三菱化
成工業(株)社製)が使用される。
スピロカルデインAまたはBスピロカルデイン
AまたはBを含む液を上記の如き吸着剤の層を通
過させて、スピロカルデインAまたはBを含む液
に含まれる不純物を吸着させて取りのぞくか、ま
たはスピロカルデインAまたはBを吸着させた
後、メタノール水、アセトン水、n−ブタノール
水などを用いて溶出することによつて得られる。
また中性脂溶性物質を培養液から採取する方法、
たとえば水と混和しない有機溶媒(たとえばクロ
ロホルム、酢酸エチル、n−ブタノールなどの単
独またはそれらの組み合せ)により培養液また
は水溶液から抽出することも可能である。更に、
スピカルデインAまたはBを含む有機溶媒層を稀
アルカリ水、稀酸性水などで洗浄することにより
混在する酸性あるいは塩基性物質を除去すること
も可能である。
このようにして得られたスピロカルデインAま
たはBを精製するためには、アビセル(旭化成工
業(株)社製)などのセルロースもしくはセフアデツ
クスLH−20(フアルマシア社製)などを用いた
分配カラムクロマトグラフイー;シリカゲル、ア
ルミナ、フロリジルのような担体を用いた吸着カ
ラムクロマトグラフイー;逆相用担体を用いた逆
相カラムクロマトグラフイー;またはスピロカル
デインAまたはBと混在する不純物との溶媒に対
する分配率の差を利用した抽出法、あるいは向流
分配法などが有効な方法といえる。以上の精製手
段を単独あるいは適宜組み合せ、反復して用いる
ことによりスピロカルデインAまたはBを精製す
ることができる。あるいは、スピロカルデインA
またはBは一般の脂溶性抗生物質と同じく、培養
条件によつては培養液中の菌体部分に存在する。
この場合はアルコール類、アセトン等の親水性有
機溶媒を用いて抽出し、抽出液より溶媒を除去
し、次いで水溶液とした後、培養液からと同様
の方法で抽出・精製することができる。
このようにして得られたスピロカルデインAは
次の理化学的および生物学的性状を有する。
(1) 物質の性状:中性の淡黄色粉末。
(2) 比旋光度:〔α〕25 D−52.7°(c,0.48,CHC
3)
(3) 分子式:C20H30O6(高分解能マススペクトル
法により測定)
(4) 分子量:366(FD/MS)
(5) 紫外線吸収スペクトル:λnaxnm(E1%
1cm)
メタノール中で測定した紫外線吸収スペクト
ルは第1図に示した通り280(sh)nm(E1%
1cm
10)に極大吸収を示す。
(6) 赤外線吸収スペクトル:νnaxcm-1
液膜法で測定した赤外線吸収スペクトルは第
2図に示す通りである。
(7) 核磁気共鳴スペクトル:δ:ppm
クロロホルム中、内部基準にTMS(テトラメ
チルシラン)を使用して測定した核磁気共鳴ス
ペクトル(400MHz)は第3図に示す通りであ
る。
(8) 溶解性:クロロホルム、酢酸エチル、アセト
ン、エタノールおよびメタノールに可溶、水に
不溶。
(9) 呈色反応:過マンガン酸カリウムおよび硫酸
に陽性。
(10) 薄層クロマトグラフイー:Rf値0.30
吸着剤;メルク社製シリカゲルプレートNo.5715
展開溶媒;酢酸エチル:ベンゼン(1:1)
(11) 抗菌スペクトル
一般グラム陽性、グラム陰性細菌に対するス
ピロカルデインAの最小発育阻止濃度(MIC)
は2%グリセリン添加感受性デイスク用寒天培
地(日水製薬(株)製)、カビ、酵母類に対しては
0.2%イーストエキス添加サブロー寒天培地、
嫌気性菌に対してはGAM寒天培地(日水製薬
(株)製)、マイコプラズマに対してはフレツシユ
イーストエキス、DNA、リン酸二カリウム、
PC−G添加PPLO寒天培地を用いた寒天培地
希釈法によつて測定した。その結果は第4〜6
表に示す通りである。
また、スピロカルデインBは次の理化学的およ
び生物学的性状を有する。
(1) 物質の性状:中性の淡黄色粉末。
(2) 比旋光度:〔α〕25 D−35.5°(c,0.62,CHC
3)
(3) 分子式:C20H32O6(高分解能マススペクトル
法により測定)
(4) 分子量:366(FD/MS)
(5) 紫外線吸収スペクトル:λnaxnm(E1%
1cm)
メタノール中で測定した紫外線吸収スペクト
ルは第4図に示した通り280(sh)nm(E1%
1cm
5)に極大吸収を示す。
(6) 赤外線吸収スペクトル:νnaxcm-1
液膜法で測定した赤外線吸収スペクトルは第
5図に示す通りである。
(7) 核磁気共鳴共鳴スペクトル:δ:ppn
重クロロホルム中、内部基準にTMS(テトラ
メチルシラン)を使用して測定した核磁気共鳴
スペクトル(400MHz)は第6図に示す通りで
ある。
(8) 溶解性:クロロホルム、酢酸エチル、アセト
ン、エタノールおよびメタノールに可溶、水に
不溶。
(9) 呈色反応:過マンガン酸カリウムおよび硫酸
に陽性。
(10) 薄層クロマトグラフイー:Rf値0.15
吸着剤;メルク社製シリカゲルプレートNo.5715
展開溶媒;酢酸エチル:ベンゼン(1:1)
(11) 抗菌スペクトル
一般グラム陽性、グラム陰性細菌に対するス
ピロカルデインBの最小発育阻止濃度(MIC)
は2%グリセリン添加感受性デイスク用寒天培
地(日水製薬(株)製)、カビ、酵母類に対しては
0.2%イーストエキス添加サブロー寒天培地、
嫌気性菌に対してはGAM寒天培地(日水製薬
(株)製)、マイコプラズマに対してはフレツシユ
イーストエキス、DNA、リン酸二カリウム、
PC−G添加PPLO寒天培地を用いた寒天培地
希釈法によつて測定した。その結果は第4〜6
表に示す通りである。[Table] +: Assimilate, ±: Weakly assimilate, -: Not assimilate
(4) Regarding intracellular components M.P.
Lechevalier et al.'s method [A.
Dietz et al., Actinomycete classification
As a result of paper chromatography analysis of the acid hydrolyzate of the bacterial cells, mesodiamino pimelic acid, arabinose, and galactose were observed, and the cell wall type was different from the cell wall type. It was confirmed that there is. The glycotype of all bacterial cells was type A. Furthermore, the method of H. Mordarska et al. [Journal of general microbiology]
Microbiology), vol. 71, p. 77, 1972], the lipids in the bacterial cells were analyzed and it was found that LCN-A
(Lipids characteristic of Nocardia) existed. Among the above-mentioned characteristics of the SANK 64282 strain, the hyphae are divided, the cell wall is type A, and the glycoform in the whole cell is type A, and there is LCN in the cell.
The presence of -A revealed that this strain belongs to the genus Nocardia in the family Nocardiaceae among actinomycetes, and was named Nocardia sp. SANK 64282. did. This strain has been deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry, and its microbial accession number is No. 6986.
This is the number. Above, we have explained spirocardin A or B producing bacteria, but it is well known that the properties of actinomycetes, especially Nocardia, are not constant and can easily change naturally or artificially. Bacteria that can be used in the present invention include all strains that produce spirocardin A or B belonging to the genus Nocardia. The culture in the present invention is carried out according to the culture method for general actinomycetes, and is preferably carried out by shaking culture in a liquid medium or by aeration stirring culture. Medium components include, for example, glucose as a carbon source,
maltose, sucrose, mannitrate, molasses,
Glycerin, dextrin, starch, soybean oil, cottonseed oil, etc. as nitrogen sources, soybean flour, peanut flour, cottonseed flour, firmamine, fishmeal, corn steep liquor, peptone, meat extract, yeast, yeast extract, sodium nitrate, Ammonium nitrate, ammonium sulfate, etc., as well as inorganic salts such as table salt,
Phosphates, calcium carbonate, trace metal salts, etc. are added as appropriate. For liquid culture,
Silicone oil, vegetable oil, surfactant, etc. are appropriately used as antifoaming agents. The pH of the medium is near neutral, and the culture temperature is 24℃ to 30℃.
℃, especially around 28℃ is preferred. Changes over time in the titer of spirocardin A or B produced over the course of the culture are shown in Table 1.
aureus) FDA 209P JC-1 as a test bacterium and a paper disk (manufactured by Toyo Kagaku Sangyo Co., Ltd., diameter 8 mm,
Thick) is measured by the assay method. Usually after 72 to 120 hours of culture, spirocardin A
Or B's production reaches its maximum value. Spirocardin A mainly present in the liquid part of the culture solution
Or B: After the completion of culture, bacterial cells and other solid parts are removed by over-operation using diatomaceous earth or the like as an aid, or by centrifugation, and extracted and purified from the liquid or supernatant. obtained by. Spirocardin A or B can be collected by utilizing its physicochemical properties, for example, using an adsorbent. Examples of adsorbents include activated carbon, adsorption resins such as Amberlite XAD-2, XAD-4, and XAD-7 (manufactured by Rohm and Haas), or Diaion HP10, HP20, HP20AG, HP50, etc. (manufactured by Mitsubishi Chemical). (manufactured by Kogyo Co., Ltd.) is used. Spirocardein A or B A liquid containing Spirocardein A or B is passed through a layer of an adsorbent as described above to adsorb and remove impurities contained in the liquid containing Spirocardein A or B, or It can be obtained by adsorbing spirocardine A or B and then eluting it using methanol water, acetone water, n-butanol water, or the like.
Also, a method for collecting neutral fat-soluble substances from the culture solution,
For example, it is also possible to extract from the culture broth or aqueous solution using an organic solvent that is immiscible with water (for example, chloroform, ethyl acetate, n-butanol, etc. alone or in combination). Furthermore,
It is also possible to remove mixed acidic or basic substances by washing the organic solvent layer containing spicaldein A or B with dilute alkaline water, dilute acidic water, or the like. In order to purify spirocardin A or B obtained in this way, partition column chromatography using cellulose such as Avicel (manufactured by Asahi Kasei Industries, Ltd.) or Sephadex LH-20 (manufactured by Pharmacia), etc. Graphite; adsorption column chromatography using a carrier such as silica gel, alumina, or florisil; reversed-phase column chromatography using a reversed-phase carrier; or to a solvent with impurities mixed with spirocardine A or B Extraction methods that utilize differences in distribution rates or countercurrent distribution methods can be said to be effective methods. Spirocardin A or B can be purified by repeatedly using the above purification means alone or in appropriate combinations. Or spirocardin A
Or B, like general lipid-soluble antibiotics, exists in the bacterial cell portion of the culture solution depending on the culture conditions.
In this case, it is possible to perform extraction using a hydrophilic organic solvent such as alcohol or acetone, remove the solvent from the extract, prepare an aqueous solution, and then extract and purify it in the same manner as from a culture solution. Spirocardin A thus obtained has the following physicochemical and biological properties. (1) Properties of substance: Neutral pale yellow powder. (2) Specific rotation: [α] 25 D −52.7° (c, 0.48, CHC
3 ) (3) Molecular formula: C 20 H 30 O 6 (measured by high-resolution mass spectrometry) (4) Molecular weight: 366 (FD/MS) (5) Ultraviolet absorption spectrum: λ nax nm (E1% 1cm) in methanol The ultraviolet absorption spectrum measured at 280 (sh) nm (E1% 1 cm
10) shows maximum absorption. (6) Infrared absorption spectrum: ν nax cm -1 The infrared absorption spectrum measured by the liquid film method is shown in Figure 2. (7) Nuclear magnetic resonance spectrum: δ: ppm The nuclear magnetic resonance spectrum (400MHz) measured in chloroform using TMS (tetramethylsilane) as an internal standard is shown in Figure 3. (8) Solubility: Soluble in chloroform, ethyl acetate, acetone, ethanol and methanol, insoluble in water. (9) Color reaction: Positive for potassium permanganate and sulfuric acid. (10) Thin layer chromatography: Rf value 0.30 Adsorbent: Merck silica gel plate No. 5715 Developing solvent: Ethyl acetate:benzene (1:1) (11) Antibacterial spectrum Spirocal against general Gram-positive and Gram-negative bacteria Minimum inhibitory concentration (MIC) of Dein A
is a 2% glycerin-added sensitive disc agar medium (manufactured by Nissui Pharmaceutical Co., Ltd.), against molds and yeasts.
Sabouraud agar medium supplemented with 0.2% yeast extract,
For anaerobic bacteria, GAM agar medium (Nissui Pharmaceutical
Co., Ltd.); for mycoplasma, fresh yeast extract, DNA, dipotassium phosphate;
It was measured by an agar medium dilution method using a PPLO agar medium supplemented with PC-G. The results are 4th to 6th
As shown in the table. In addition, spirocardin B has the following physicochemical and biological properties. (1) Properties of substance: Neutral pale yellow powder. (2) Specific rotation: [α] 25 D −35.5° (c, 0.62, CHC
3 ) (3) Molecular formula: C 20 H 32 O 6 (measured by high-resolution mass spectrometry) (4) Molecular weight: 366 (FD/MS) (5) Ultraviolet absorption spectrum: λ nax nm (E1% 1cm) in methanol The ultraviolet absorption spectrum measured at 280 (sh) nm (E1% 1 cm
5) shows maximum absorption. (6) Infrared absorption spectrum: ν nax cm -1 The infrared absorption spectrum measured by the liquid film method is shown in FIG. (7) Nuclear magnetic resonance spectrum: δ: The nuclear magnetic resonance spectrum (400 MHz) measured in ppn deuterated chloroform using TMS (tetramethylsilane) as an internal standard is shown in FIG. (8) Solubility: Soluble in chloroform, ethyl acetate, acetone, ethanol and methanol, insoluble in water. (9) Color reaction: Positive for potassium permanganate and sulfuric acid. (10) Thin layer chromatography: Rf value 0.15 Adsorbent: Merck silica gel plate No. 5715 Developing solvent: Ethyl acetate:benzene (1:1) (11) Antibacterial spectrum Spirocal against general Gram-positive and Gram-negative bacteria Minimum inhibitory concentration (MIC) of Dein B
is a 2% glycerin-added sensitive disc agar medium (manufactured by Nissui Pharmaceutical Co., Ltd.), against molds and yeasts.
Sabouraud agar medium supplemented with 0.2% yeast extract,
For anaerobic bacteria, GAM agar medium (Nissui Pharmaceutical
Co., Ltd.); for mycoplasma, fresh yeast extract, DNA, dipotassium phosphate;
It was measured by an agar medium dilution method using a PPLO agar medium supplemented with PC-G. The results are 4th to 6th
As shown in the table.
【表】【table】
【表】【table】
【表】
イフイシル
[Table] Ifisil
【表】【table】
【表】
上記の如きスピロカルデインAまたはBの特性
を既知抗生物質の諸性状と比較した結果、一致す
るものはなく新抗生物質と判定された。
以上から、スピロカルデインAまたはBは各種
細菌感染性疾患を対照とする抗菌剤として使用さ
れる。その投与形態としては皮下注射、静脈内注
射、筋肉注射、坐剤などによる非経口投与法ある
いは錠剤、カプセル剤、酸剤、顆粒剤などによる
経口投与法があげられる。投与量は対象疾患、投
与経路および投与回数などによつて異なるが、例
えば成人に対して通常は1日500mg乃至2000mgを
1回または数回に分けて投与するのが好ましい。
次に実施例をあげて本発明をさらに具体的に説
明する。
実施例 1
ノカルジアSP.SANK64282株を培地組成−1
で示される培地80mlを含む500ml容三角フラスコ
に一白金耳接種し、220rpmの回転振盪培養機に
より28℃で96時間培養した。この培養液25mlを培
地組成−2で示される培地500mlを含む2容三
角フラスコ4本に接種し、220rpmの回転振盪培
養機により28℃で24時間培養した。得られた培養
液を同一の培地15を含む30容ジヤーフアーメ
ンター2基に各々750mlずつ接種し、28℃、撹拌
150回転/分、通気量10/分で67時間通気撹拌
培養した。得られた培養液30に過助剤として
セライト545(米国ジヨンズ・マンビル・プロダク
ト・コーポレーシヨン製)を1.5Kg加えて過す
ると、液30(PH7.1)が得られた。この培養
液を15の酢酸エチルで2回抽出し、30の飽
和食塩水で洗浄後、無水硫酸ナトリウムで脱水
し、源圧下で濃縮乾固すると1.2の油状物が得
られた。この油状物をベンゼンに溶解し、あらか
じめベンゼンで調製したシリカゲルカラム(米国
マリンクロツト社製、シリカゲル10g)に吸着さ
せ、ベンゼンで洗浄後、順次、ベンゼン:酢酸エ
チルの3:1、1:1、1:2の混合溶媒で20ml
ずつ分画し展開・溶出した。スピロカルデインA
はフラクシヨンNo.6〜9に溶出され、スピロカル
デインBはフラクシヨンNo.10〜13に溶出された。
それぞれを源圧下で濃縮乾固すると、スピロカル
デインAが油状物として260mg、スピロカルデイ
ンBが油状物として235mg得られた。得られた粗
スピロカルデインAは再度シリカゲルカラム(10
ml)に吸着させ各々60mlのベンゼン、クロロホル
ム、5%アセトン−クロロホルムの混合溶媒で展
開し、5mlずつ分画してフラクシヨンNo.36まで溶
出した。スピロカルデインAはフラクシヨンNo.15
〜24に溶出された。活性分画を併せ減圧下で濃縮
乾燥固することにより27mgのスピロカルデインA
が得られた。一方、得られた粗スピロカルデイン
Bは再度シリカゲルカラム(10g)に吸着させ
各々50mlのベンゼン、ベンゼン:酢酸エチルの
3:1、2:1、1:1、1:2の混合溶媒で展
開し、7mlずつ分画してフラクシヨンNo.43まで溶
出した。スピロカルデインBはフラクシヨンNo.19
〜31に溶出された。活性分画を併せ減圧下で濃縮
乾固することにより80mgのスピロカルデインBが
得られた。
培地組成−1
グルコース 1%
グリセリン 1%
シユクロース 1%
生イースト 1%
大豆粉 2%
オートミール 0.5%
カザミノ酸 0.5%
炭酸カルシウム 0.1%
ニツサンデイスホームCB−442(消泡剤)PH7.0
0.01%
培地組成−2
グルコース 2%
ラスターゲンFK 1%
生イースト 0.9%
肉エキス 0.5%
ポリペプトン 0.5%
炭酸カルシウム 0.3%
塩化ナトリウム 0.5%
ニツサンデイスホームCB−442(消泡剤)PH7.0
0.01%
実施例 2
ノカルジアSP.SANK64282株を培地組成−1
で示される培地80mlを含む500ml容三角フラスコ
に一白金耳接種し、200rpmの回転振盪培養機に
より28℃で96時間培養した。この培養液25mlを培
地組成−2で示される培地500mlを含む2容三
角フラスコ2本に接種し、220rpmの回転振盪培
養機により28℃で48時間培養した。得られた培養
液750mlを培地組成−2で示される培地15を含
む30容ジヤーフアーメンターに接種し、28℃で
24時間培養した。得られた培養液15を同一培地
300を含む600容タンクに接種し、28℃、撹拌
100回転/分、通気量300/分で115時間通気撹
拌培養した。得られた培養液300に過助剤と
してセライト545を15Kg加えて過すると、液
290が得られた。この培養液を160の酢酸エ
チルで2回抽出し、減圧下で濃縮乾固すると43g
の油状物が得られた。この油状物をn−ヘキサン
に溶解し、あらかじめn−ヘキサンで調製したシ
リカゲルカラム(米国マリンクロツト社製、シリ
カゲル250g)に吸着させ、各々500mlの5,10,
20,30,40,50%アセトンを含むn−ヘキサン溶
液の混合溶媒で20mlずつ分画し展開・溶出した。
スピロカルデインAはフラクシヨンNo.54〜85に溶
出され、スピロカルデインBはフラクシヨンNo.86
〜100に溶出された。それぞれを減圧下で濃縮乾
固するとスピロカルデインAが7.6g、スピロカ
ルデインBが4.5gそれぞれ油状物として得られ
た。得られた粗スピカルデインA7.6gは再度シ
リカゲルカラム(50g)に吸着させ各々300mlの
ベンゼン:酢酸エチルの4:1、3:1、2:
1、1:1の混合溶媒で展開し、20mlずつ分画し
て溶出した。フラクシヨンNo.14〜44をあつめ、減
圧下で濃縮乾固すると、スピロカルデインA2.4
gの油状物がられた。一方、得られた粗スピロカ
ルデインB4.1gも同様に再度シリカゲルカラム
(50g)によるクロマトに付した。各々300mlのベ
ンゼン:酢酸エチルの3:1、2:1、1:1、
1:2の混合溶媒で展開し20mlずつ分画して溶出
した。フラクシヨンNo.45〜60をあつめ、減圧下で
濃縮乾固すると、スピロカルデインB471mgの油
状物が得られた。得られたスピロカルデインAま
たはBを逆相クロマトグラフイー(メルク社製、
ローバー・カラムリクロプレツプRP−8(サイズ
B))に付して更に精製した。スピロカルデイン
A2.4gを4mlのメタノールに溶解し、そのうち
1mlをあらかじめ60%メタノール水で調製したロ
ーバー・カラムリクロプレツプRP−8(サイズ
B)に吸着後、屈折計を用いて溶出液の屈折率を
調べながら60%メタノール水で展開、溶出した。
スピロカルデインAはサンプル吸着後30分より36
分の間に溶出され、活性分画として60mlが得られ
た。更に残る3mlのサンプルについて同様の操作
を3回くりかえすと、活性分画として合計180ml
が得られた。得られた活性分画より、減圧下でメ
タノールを留去し、100mlの酢酸エチルで抽出し、
抽出液を飽和食塩水で洗浄後、芒硝で脱水し濃縮
乾固することにより黄色粉末のスピロカルデイン
Aが170mg得られた。同様に、スピロカルデイン
B470mgを2mlのメタノールに溶解しスピロカル
デインAと同一条件で同様の操作を行つた。スピ
ロカルデインBはサンプル吸着後33分より43分の
間に溶出され活性分画として110mlが得られた。
残る1mlのサンプルについても同様の操作をくり
かえすと、活性分画として110mlが得られた。得
られた活性分画より、減圧下でメタノールを留去
し、100mlの酢酸エチルで抽出し、抽出液を飽和
食塩水で洗浄後、芒硝で脱水し濃縮乾固すること
により黄色粉末のスピロカルデインBが190mg得
られた。
本実施例において使用された培地組成−1およ
び2は、実施例1で使用されたものと同じであ
る。[Table] As a result of comparing the properties of spirocardin A or B as described above with those of known antibiotics, there was no match and it was determined that it is a new antibiotic. From the above, spirocardin A or B is used as an antibacterial agent to control various bacterial infectious diseases. The administration form includes parenteral administration such as subcutaneous injection, intravenous injection, intramuscular injection, and suppository, and oral administration using tablets, capsules, acid preparations, granules, and the like. The dosage varies depending on the target disease, route of administration, frequency of administration, etc., but for adults, it is usually preferable to administer 500 mg to 2000 mg per day, either once or in divided doses. Next, the present invention will be explained in more detail with reference to Examples. Example 1 Culture medium composition-1 for Nocardia SP.SANK64282 strain
One platinum loop was inoculated into a 500 ml Erlenmeyer flask containing 80 ml of the medium shown in , and cultured at 28°C for 96 hours in a rotary shaking incubator at 220 rpm. 25 ml of this culture solution was inoculated into four 2-volume Erlenmeyer flasks containing 500 ml of the medium shown in medium composition-2, and cultured at 28° C. for 24 hours in a rotary shaking incubator at 220 rpm. Inoculate 750 ml of the obtained culture solution into two 30-volume jar fermenters each containing 15 of the same medium, and stir at 28°C.
Aeration and agitation culture was carried out for 67 hours at 150 revolutions/min and aeration rate of 10/min. When 1.5 kg of Celite 545 (manufactured by John's Manville Products Corporation, USA) was added as a supernatant to the obtained culture solution 30 and filtered, solution 30 (PH 7.1) was obtained. This culture solution was extracted twice with 15 portions of ethyl acetate, washed with 30 portions of saturated brine, dehydrated with anhydrous sodium sulfate, and concentrated to dryness under source pressure to obtain 1.2 oily substance. This oil was dissolved in benzene and adsorbed on a silica gel column (manufactured by Mallinckrodt, USA, 10 g of silica gel) prepared in advance with benzene. After washing with benzene, benzene:ethyl acetate was applied in sequence at 3:1, 1:1, and 1. :20ml of mixed solvent of 2
It was fractionated, developed and eluted. Spirocardine A
was eluted in fractions No. 6 to 9, and spirocardine B was eluted in fractions No. 10 to 13.
Each was concentrated to dryness under source pressure to obtain 260 mg of spirocardein A as an oil and 235 mg of spirocardein B as an oil. The obtained crude spirocardin A was again passed through a silica gel column (10
ml) and developed with 60 ml of a mixed solvent of benzene, chloroform, and 5% acetone-chloroform, fractionated into 5 ml portions, and eluted up to fraction No. 36. Spirocaldein A is fraction No.15
eluted at ~24. By combining the active fractions and concentrating to dryness under reduced pressure, 27 mg of spirocardin A was obtained.
was gotten. On the other hand, the obtained crude spirocardine B was adsorbed onto a silica gel column (10 g) again and developed with 50 ml of each benzene and a mixed solvent of benzene:ethyl acetate of 3:1, 2:1, 1:1, and 1:2. The mixture was fractionated into 7 ml portions and fraction No. 43 was eluted. Spirocaldein B is fraction No.19
eluted at ~31. The active fractions were combined and concentrated to dryness under reduced pressure to obtain 80 mg of spirocardin B. Medium composition-1 Glucose 1% Glycerin 1% Sucrose 1% Fresh yeast 1% Soybean flour 2% Oatmeal 0.5% Casamino acids 0.5% Calcium carbonate 0.1% Nitsusan Daisu Home CB-442 (antifoaming agent) PH7.0
0.01% Medium composition-2 Glucose 2% Lastagen FK 1% Fresh yeast 0.9% Meat extract 0.5% Polypeptone 0.5% Calcium carbonate 0.3% Sodium chloride 0.5% Nitsusan Days Home CB-442 (antifoaming agent) PH7.0
0.01% Example 2 Nocardia SP.SANK64282 strain in medium composition-1
One platinum loop was inoculated into a 500 ml Erlenmeyer flask containing 80 ml of the medium shown in , and cultured at 28°C for 96 hours in a rotary shaking incubator at 200 rpm. 25 ml of this culture solution was inoculated into two 2-volume Erlenmeyer flasks containing 500 ml of the medium shown in Medium Composition-2, and cultured at 28°C for 48 hours in a rotary shaking incubator at 220 rpm. 750 ml of the obtained culture solution was inoculated into a 30-volume jar fermenter containing medium 15 shown in medium composition-2, and incubated at 28°C.
Cultured for 24 hours. The obtained culture solution 15 was added to the same medium.
Inoculate a 600 volume tank containing 300, stir at 28℃
Aeration and agitation culture was carried out for 115 hours at 100 revolutions/min and aeration rate of 300/min. When 15kg of Celite 545 was added as a supernatant to the obtained culture solution 300 and filtered, the solution
290 was obtained. This culture solution was extracted twice with 160 ml of ethyl acetate and concentrated to dryness under reduced pressure, yielding 43 g.
of oil was obtained. This oil was dissolved in n-hexane and adsorbed on a silica gel column (manufactured by Mallinckrodt Co., USA, 250 g of silica gel) prepared in advance with n-hexane.
The mixture was fractionated into 20 ml portions using a mixed solvent of n-hexane solutions containing 20, 30, 40, and 50% acetone, and developed and eluted.
Spirocardein A is eluted in fractions No. 54 to 85, and spirocardein B is eluted in fraction No. 86.
eluted at ~100. When each was concentrated to dryness under reduced pressure, 7.6 g of spirocardein A and 4.5 g of spirocardein B were obtained as oils. The obtained crude spicaldein A (7.6 g) was again adsorbed onto a silica gel column (50 g) and mixed with 300 ml of benzene:ethyl acetate (4:1, 3:1, 2:3).
The mixture was developed with a mixed solvent of 1:1 and eluted in fractions of 20 ml. Fractions No. 14 to 44 were collected and concentrated to dryness under reduced pressure to produce spirocardine A2.4.
g of oily substance was removed. On the other hand, 4.1 g of the obtained crude spirocardin B was similarly subjected to chromatography again using a silica gel column (50 g). 300ml each of benzene:ethyl acetate 3:1, 2:1, 1:1,
It was developed with a 1:2 mixed solvent, fractionated into 20 ml portions, and eluted. Fractions Nos. 45 to 60 were collected and concentrated to dryness under reduced pressure to obtain 471 mg of spirocardine B as an oil. The obtained spirocardin A or B was subjected to reverse phase chromatography (manufactured by Merck & Co., Ltd.,
Further purification was performed using Rover Column Ricroprep RP-8 (size B). spirocardine
Dissolve 2.4 g of A in 4 ml of methanol, adsorb 1 ml of it onto Rover Column Ricroprep RP-8 (size B) prepared in advance with 60% methanol water, and then measure the refraction of the eluate using a refractometer. It was developed and eluted with 60% methanol water while checking the ratio.
Spirocardin A starts from 30 minutes after sample adsorption36
The active fraction was eluted in 60 ml. Repeating the same procedure three times for the remaining 3 ml of sample yields a total of 180 ml of active fraction.
was gotten. Methanol was distilled off from the obtained active fraction under reduced pressure, extracted with 100 ml of ethyl acetate,
The extract was washed with saturated brine, dehydrated with sodium sulfate, and concentrated to dryness to obtain 170 mg of spirocardin A as a yellow powder. Similarly, spirocardine
470 mg of B was dissolved in 2 ml of methanol and the same operation as for spirocardin A was carried out under the same conditions. Spirocardin B was eluted between 33 and 43 minutes after sample adsorption, and 110 ml of active fraction was obtained.
When the same operation was repeated for the remaining 1 ml of sample, 110 ml of active fraction was obtained. From the obtained active fraction, methanol was distilled off under reduced pressure, extracted with 100 ml of ethyl acetate, the extract was washed with saturated saline, dehydrated with Glauber's salt, and concentrated to dryness to obtain spirocal as a yellow powder. 190 mg of Dein B was obtained. Medium compositions-1 and 2 used in this example are the same as those used in Example 1.
第1図はスピロカルデインAの紫外線吸収スペ
クトルを示し、第2図は同物質の赤外線吸収スペ
クトルを示し、第3図は同物質の核磁気共鳴スペ
クトルを示す。第4図はスピロカルデインBの紫
外線吸収スペクトルを示し、第5図は同物質の赤
外線吸収スペクトルを示し、第6図は同物質の核
磁気共鳴スペクトルを示す。
FIG. 1 shows the ultraviolet absorption spectrum of spirocardine A, FIG. 2 shows the infrared absorption spectrum of the same substance, and FIG. 3 shows the nuclear magnetic resonance spectrum of the same substance. FIG. 4 shows the ultraviolet absorption spectrum of spirocardine B, FIG. 5 shows the infrared absorption spectrum of the same substance, and FIG. 6 shows the nuclear magnetic resonance spectrum of the same substance.
Claims (1)
たはB生産菌を培養してその培養物よりスピロカ
ルデインAまたはBを採取することよりなるスピ
ロカルデインAまたはBの製造法。 3 ノカルジア属に属するスピロカルデインAま
たはB生産菌がノカルジア sp.SANK 64282
株(微工研菌寄第6986号)である特許請求の範囲
第2項記載の製造法。[Claims] 1 formula Spirocardin A or B with However, in the formula, in spirocardine A, X represents a group [formula], and in spirocardine B, X represents a group [formula]. 2. A method for producing spirocardin A or B, which comprises culturing spirocardin A or B-producing bacteria belonging to the genus Nocardia and collecting spirocardin A or B from the culture. 3 The spirocardin A or B producing bacterium belonging to the genus Nocardia is Nocardia sp.SANK 64282
2. The production method according to claim 2, which is a strain (KAIKOKEN HYUKYO NO. 6986).
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP58234037A JPS60126089A (en) | 1983-12-12 | 1983-12-12 | Antibiotic substance spirocardin a and b and their production |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP58234037A JPS60126089A (en) | 1983-12-12 | 1983-12-12 | Antibiotic substance spirocardin a and b and their production |
Publications (2)
Publication Number | Publication Date |
---|---|
JPS60126089A JPS60126089A (en) | 1985-07-05 |
JPH0445516B2 true JPH0445516B2 (en) | 1992-07-27 |
Family
ID=16964570
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP58234037A Granted JPS60126089A (en) | 1983-12-12 | 1983-12-12 | Antibiotic substance spirocardin a and b and their production |
Country Status (1)
Country | Link |
---|---|
JP (1) | JPS60126089A (en) |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS5745415A (en) * | 1980-09-02 | 1982-03-15 | Tokyo Electric Co Ltd | Measuring label issuer |
-
1983
- 1983-12-12 JP JP58234037A patent/JPS60126089A/en active Granted
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS5745415A (en) * | 1980-09-02 | 1982-03-15 | Tokyo Electric Co Ltd | Measuring label issuer |
Also Published As
Publication number | Publication date |
---|---|
JPS60126089A (en) | 1985-07-05 |
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