NO744292L - - Google Patents
Info
- Publication number
- NO744292L NO744292L NO744292A NO744292A NO744292L NO 744292 L NO744292 L NO 744292L NO 744292 A NO744292 A NO 744292A NO 744292 A NO744292 A NO 744292A NO 744292 L NO744292 L NO 744292L
- Authority
- NO
- Norway
- Prior art keywords
- methanol
- new
- strain
- grams
- liters
- Prior art date
Links
- 239000003242 anti bacterial agent Substances 0.000 claims description 17
- 229940088710 antibiotic agent Drugs 0.000 claims description 17
- 235000015097 nutrients Nutrition 0.000 claims description 14
- 238000000034 method Methods 0.000 claims description 11
- 235000001674 Agaricus brunnescens Nutrition 0.000 claims description 8
- 241000123975 Trichoderma polysporum Species 0.000 claims description 8
- 241000875119 Cylindrocarpon lucidum Species 0.000 claims description 5
- 238000004519 manufacturing process Methods 0.000 claims description 4
- 230000002538 fungal effect Effects 0.000 claims 1
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 66
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 22
- 239000000243 solution Substances 0.000 description 22
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 14
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 13
- 239000000203 mixture Substances 0.000 description 13
- 239000000741 silica gel Substances 0.000 description 13
- 229910002027 silica gel Inorganic materials 0.000 description 13
- WSLDOOZREJYCGB-UHFFFAOYSA-N 1,2-Dichloroethane Chemical compound ClCCCl WSLDOOZREJYCGB-UHFFFAOYSA-N 0.000 description 12
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 12
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 12
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 12
- 239000000126 substance Substances 0.000 description 12
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 9
- 241000228245 Aspergillus niger Species 0.000 description 8
- 239000000725 suspension Substances 0.000 description 8
- 229920001817 Agar Polymers 0.000 description 7
- 239000008272 agar Substances 0.000 description 7
- 238000004587 chromatography analysis Methods 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 239000003480 eluent Substances 0.000 description 7
- 239000000523 sample Substances 0.000 description 7
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 6
- 230000003115 biocidal effect Effects 0.000 description 6
- 239000000284 extract Substances 0.000 description 6
- 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 6
- 239000003208 petroleum Substances 0.000 description 6
- 239000002904 solvent Substances 0.000 description 6
- 229910000831 Steel Inorganic materials 0.000 description 5
- 238000000855 fermentation Methods 0.000 description 5
- 230000004151 fermentation Effects 0.000 description 5
- 150000002500 ions Chemical class 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 239000010959 steel Substances 0.000 description 5
- WHBHBVVOGNECLV-OBQKJFGGSA-N 11-deoxycortisol Chemical compound O=C1CC[C@]2(C)[C@H]3CC[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 WHBHBVVOGNECLV-OBQKJFGGSA-N 0.000 description 4
- 241000699670 Mus sp. Species 0.000 description 4
- 229910052799 carbon Inorganic materials 0.000 description 4
- 150000001875 compounds Chemical class 0.000 description 4
- 239000011888 foil Substances 0.000 description 4
- TWNQGVIAIRXVLR-UHFFFAOYSA-N oxo(oxoalumanyloxy)alumane Chemical compound O=[Al]O[Al]=O TWNQGVIAIRXVLR-UHFFFAOYSA-N 0.000 description 4
- 241000894007 species Species 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 229930182555 Penicillin Natural products 0.000 description 3
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 3
- 241000700159 Rattus Species 0.000 description 3
- 229930006000 Sucrose Natural products 0.000 description 3
- 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 3
- 238000005273 aeration Methods 0.000 description 3
- 206010003246 arthritis Diseases 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000010828 elution Methods 0.000 description 3
- 238000001704 evaporation Methods 0.000 description 3
- 230000008020 evaporation Effects 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 230000001506 immunosuppresive effect Effects 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 229940049954 penicillin Drugs 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 239000005720 sucrose Substances 0.000 description 3
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- DKPFZGUDAPQIHT-UHFFFAOYSA-N Butyl acetate Natural products CCCCOC(C)=O DKPFZGUDAPQIHT-UHFFFAOYSA-N 0.000 description 2
- 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 2
- 206010018910 Haemolysis Diseases 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 241000364057 Peoria Species 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
- 239000013543 active substance Substances 0.000 description 2
- 239000003463 adsorbent Substances 0.000 description 2
- 230000000274 adsorptive effect Effects 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 239000008346 aqueous phase Substances 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
- 210000001185 bone marrow Anatomy 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000000921 elemental analysis Methods 0.000 description 2
- 230000008030 elimination Effects 0.000 description 2
- 238000003379 elimination reaction Methods 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 230000008588 hemolysis Effects 0.000 description 2
- KDCIHNCMPUBDKT-UHFFFAOYSA-N hexane;propan-2-one Chemical compound CC(C)=O.CCCCCC KDCIHNCMPUBDKT-UHFFFAOYSA-N 0.000 description 2
- FUZZWVXGSFPDMH-UHFFFAOYSA-N hexanoic acid Chemical compound CCCCCC(O)=O FUZZWVXGSFPDMH-UHFFFAOYSA-N 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 239000011630 iodine Substances 0.000 description 2
- 229910052740 iodine Inorganic materials 0.000 description 2
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 2
- 238000001819 mass spectrum Methods 0.000 description 2
- 230000008018 melting Effects 0.000 description 2
- 238000002844 melting Methods 0.000 description 2
- 239000012074 organic phase Substances 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 239000001965 potato dextrose agar Substances 0.000 description 2
- 238000011321 prophylaxis Methods 0.000 description 2
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000012827 research and development Methods 0.000 description 2
- FSYKKLYZXJSNPZ-UHFFFAOYSA-N sarcosine Chemical compound C[NH2+]CC([O-])=O FSYKKLYZXJSNPZ-UHFFFAOYSA-N 0.000 description 2
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000007858 starting material Substances 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 238000011282 treatment Methods 0.000 description 2
- SJHPCNCNNSSLPL-CSKARUKUSA-N (4e)-4-(ethoxymethylidene)-2-phenyl-1,3-oxazol-5-one Chemical compound O1C(=O)C(=C/OCC)\N=C1C1=CC=CC=C1 SJHPCNCNNSSLPL-CSKARUKUSA-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
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 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
- QWCKQJZIFLGMSD-UHFFFAOYSA-N 2-Aminobutanoic acid Natural products CCC(N)C(O)=O QWCKQJZIFLGMSD-UHFFFAOYSA-N 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 208000032116 Autoimmune Experimental Encephalomyelitis Diseases 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 229920002261 Corn starch Polymers 0.000 description 1
- 241000723247 Cylindrocarpon Species 0.000 description 1
- QWCKQJZIFLGMSD-GSVOUGTGSA-N D-alpha-aminobutyric acid Chemical compound CC[C@@H](N)C(O)=O QWCKQJZIFLGMSD-GSVOUGTGSA-N 0.000 description 1
- 208000009386 Experimental Arthritis Diseases 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 239000006000 Garlic extract Substances 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 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
- 241000124008 Mammalia Species 0.000 description 1
- 206010033661 Pancytopenia Diseases 0.000 description 1
- 206010033799 Paralysis Diseases 0.000 description 1
- 208000025747 Rheumatic disease Diseases 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 1
- 108010077895 Sarcosine Proteins 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- 241000223259 Trichoderma Species 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 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
- 230000001133 acceleration Effects 0.000 description 1
- 238000013019 agitation Methods 0.000 description 1
- 235000004279 alanine Nutrition 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
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000002579 anti-swelling effect Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-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
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000000973 chemotherapeutic effect Effects 0.000 description 1
- WORJEOGGNQDSOE-UHFFFAOYSA-N chloroform;methanol Chemical compound OC.ClC(Cl)Cl WORJEOGGNQDSOE-UHFFFAOYSA-N 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 239000013068 control sample Substances 0.000 description 1
- 235000005822 corn Nutrition 0.000 description 1
- 239000008120 corn starch Substances 0.000 description 1
- 208000024389 cytopenia Diseases 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 238000005238 degreasing Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- IJKVHSBPTUYDLN-UHFFFAOYSA-N dihydroxy(oxo)silane Chemical compound O[Si](O)=O IJKVHSBPTUYDLN-UHFFFAOYSA-N 0.000 description 1
- AIUDWMLXCFRVDR-UHFFFAOYSA-N dimethyl 2-(3-ethyl-3-methylpentyl)propanedioate Chemical compound CCC(C)(CC)CCC(C(=O)OC)C(=O)OC AIUDWMLXCFRVDR-UHFFFAOYSA-N 0.000 description 1
- 229910000396 dipotassium phosphate Inorganic materials 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 201000002491 encephalomyelitis Diseases 0.000 description 1
- 210000003743 erythrocyte Anatomy 0.000 description 1
- OAMZXMDZZWGPMH-UHFFFAOYSA-N ethyl acetate;toluene Chemical compound CCOC(C)=O.CC1=CC=CC=C1 OAMZXMDZZWGPMH-UHFFFAOYSA-N 0.000 description 1
- 235000020706 garlic extract Nutrition 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 230000035931 haemagglutination Effects 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 229940027941 immunoglobulin g Drugs 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 201000002364 leukopenia Diseases 0.000 description 1
- 231100001022 leukopenia Toxicity 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 239000000401 methanolic extract Substances 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- XJODGRWDFZVTKW-ZCFIWIBFSA-N n-methylleucine Chemical compound CN[C@@H](C(O)=O)CC(C)C XJODGRWDFZVTKW-ZCFIWIBFSA-N 0.000 description 1
- 210000000944 nerve tissue Anatomy 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 238000005191 phase separation Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 108010009004 proteose-peptone Proteins 0.000 description 1
- 239000013558 reference substance Substances 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000004317 sodium nitrate Substances 0.000 description 1
- 235000010344 sodium nitrate Nutrition 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
- 230000000451 tissue damage Effects 0.000 description 1
- 231100000827 tissue damage Toxicity 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 239000002023 wood Substances 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
- 239000001052 yellow pigment Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K4/00—Peptides having up to 20 amino acids in an undefined or only partially defined sequence; Derivatives thereof
- C07K4/06—Peptides having up to 20 amino acids in an undefined or only partially defined sequence; Derivatives thereof from fungi
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/06—Fungi, e.g. yeasts
- A61K36/062—Ascomycota
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/885—Trichoderma
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Medicinal Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Molecular Biology (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Pharmacology & Pharmacy (AREA)
- Botany (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Medical Informatics (AREA)
- Biomedical Technology (AREA)
- Alternative & Traditional Medicine (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Biophysics (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Epidemiology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
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Description
Foreliggende oppfinnelse vedrorer en fremgangsmåte for fremstilling av de nye antibiotika S 7481/F-l og S 7481/F-2. The present invention relates to a method for the production of the new antibiotics S 7481/F-1 and S 7481/F-2.
Det særegne ved fremgangsmåten i henhold til oppfinnelsen for fremstilling av de nye antibiotika S 7481/F-l og S 7481/F-2 er at en stamme av sopparten Cylindrocarpon lucidum Booth henhv. sopparten Trichoderma polysporum (Link ex Pers.) Rifai dyrkes på The peculiarity of the method according to the invention for the production of the new antibiotics S 7481/F-1 and S 7481/F-2 is that a strain of the mushroom species Cylindrocarpon lucidum Booth respectively. the fungus Trichoderma polysporum (Link ex Pers.) Rifai is grown on
eller i et næringsmedium, de nye antibiotika isoleres fra gjæringsbuljongen på i og for seg kjent måte ved ekstraktive og/eller adsorptive arbeidsmetoder og renses deretter kromatografisk eller ved hjelp av motstromsfordeling. or in a nutrient medium, the new antibiotics are isolated from the fermentation broth in a manner known per se by extractive and/or adsorptive working methods and then purified chromatographically or by means of countercurrent distribution.
Den nye i henhold til oppfinnelsen anvendte stamme av sopparten Cylindrocarpon lucidum Booth ble isolert fra en jordprove som ble funnet i Wisconsin, USA og en kultur derav ble deponert i United States Department of Agriculture (Northers Research and Development Division), Peoria, 111., USA under nummer NRRL 5760. The new strain of the fungus Cylindrocarpon lucidum Booth used according to the invention was isolated from a soil sample found in Wisconsin, USA and a culture thereof was deposited in the United States Department of Agriculture (Northers Research and Development Division), Peoria, 111., USA under number NRRL 5760.
Den nye stamme NRRL 5760 av Cylindrocarpon lucidum Booth stemmerThe new strain NRRL 5760 of Cylindrocarpon lucidum Booth agrees
i vesentlige trekk overens med orginalbeskrivelsen (C. Booth 1966; largely consistent with the original description (C. Booth 1966;
The Genus Cylindrocarpon; Mycological Paper nr. 104, 21). På potet-dextrose-agar danner stammen NRRL 5760 hverken klamdysporer eller mikrokonidier. Klamdysporaktive celler i makrokonidiene mangler likeledes. De bananformede, oftest med tre tverrvegger oppdelte makrokénidier er 28 - 45 x 4,8 - 5,7^u store og er dermed betydelig mindre enn angitt i den ovennevnte beskrivelse (45 - 65 x 6 - 6,5^u). En ytterligere viktig forskjell består i vekst-hastigheten, idet mens denne Typus i lopet av 7 dager skulle danne 1 - 1,5 cm store kolonier, vokser stammen NRRL 5760 i lopet av den samme tid til 7 - 8 cm store kolonier, idet det på potet-dextroseagar nesten utelukkende dannes fargelost til brunt substratmycel. Konidioforene med phialidene dannes allerede etter få dager i tette grupper og knipper, forgrener seg penicillium-til verticiliumaktig og danner i lopet av lengre tid makrokonidier som samler seg som en slimet, beige masse. The Genus Cylindrocarpon; Mycological Paper No. 104, 21). On potato dextrose agar, the strain NRRL 5760 forms neither sticky dysspores nor microconidia. Chlamydiaspore-active cells in the macroconidia are likewise lacking. The banana-shaped macrokenidia, usually divided by three transverse walls, are 28 - 45 x 4.8 - 5.7^u in size and are thus significantly smaller than indicated in the above description (45 - 65 x 6 - 6.5^u). A further important difference consists in the growth rate, as while this Typus should form 1 - 1.5 cm large colonies in the course of 7 days, the strain NRRL 5760 grows in the course of the same time to 7 - 8 cm large colonies, as on potato-dextrose agar almost exclusively colorless to brown substrate mycelium is formed. The conidiophores with the phialides form already after a few days in dense groups and bunches, branch penicillium- to verticillium-like and over time form macroconidia that collect as a slimy, beige mass.
Den nye i henhold til oppfinnelsen anvendte stamme av sopparten Trichoderma polysporum ble isolert fra en i Norge funnet jordprove og en kultur derav ble deponert i United States Department of Agriculture (Northern Research and Development Division), Peoria, 111., USA under nr. NRRL 8044. The new strain of the fungus species Trichoderma polysporum used in accordance with the invention was isolated from a soil sample found in Norway and a culture thereof was deposited in the United States Department of Agriculture (Northern Research and Development Division), Peoria, 111., USA under no. NRRL 8044.
Den nye stamme NRRL 8044 av Trichoderma polysporum (Link ex Pers). Rifai vokser på 2% maltekstraktagar ved 6 til 33°C og vekst-optimum ligger ved 24°C, idet det i lopet av 12 dager dannes rent hvite, på overflaten floyelsaktige og ufoldede kolonier med tverrmål 40 - 50 mm. Ved den hoyeste temperatur som enda tillater god vekst (33°C) dannes derimot over hodet ikke noe luftmycel, men derimot en meget sterkt foldet koloni. Med tiltagende dyrkingstemperatur dannes på koloniens underside et voksende gult pigment som ved 27°C og mer utpreget ved 30 - 33°C diffunderer inn i agaren. The new strain NRRL 8044 of Trichoderma polysporum (Link ex Pers). Rifai grows on 2% malt extract agar at 6 to 33°C and the growth optimum is at 24°C, as pure white, velvety on the surface and unfolded colonies with a transverse dimension of 40 - 50 mm are formed in the course of 12 days. At the highest temperature that still allows good growth (33°C), on the other hand, no aerial mycelium is formed above the head, but instead a very strongly folded colony. With increasing cultivation temperature, a growing yellow pigment forms on the underside of the colony, which diffuses into the agar at 27°C and more pronouncedly at 30 - 33°C.
For dannelse av konidioforene, som både står spredt, men også i tettere grupper, fremfor alt på dyrkingskar-kanten, trenger den nye stamme av Trichoderma polysporum ved 24°C på maltekstraktagar 15 - 20 dager. Morfologien av bifruktformen avviker i noen trekk litt fra beskrivelsen for denne art (M.A. Rifai 1969: A REVISION OF THE GENUS TRICHODERMA in Mycological Papers, No. 116). Således når hovedgrenen av konidiebærerne bare et tverrmål på 2,0 - 3,8^a, mens dét for arten er angitt 4 6,3 ^u. Phialidene i den nye stamme av Trichoderma polysporum er, spesielt ved slutten av deres utvikling, strukket i lengden og dette er typisk for denne art. Til slutt er konidiene noe mindre enn angitt i artsbeskrivelsen, nemlig 2,0 - 3,1 x 1,5 - 2,0 i forhold til 2,4 - 3,8 x 1,8 2,2^1. Den foreliggende Trichoderma polysporum stamme NRRL 8044 danner under alle de nevnte dyrkingsbetingelser The new strain of Trichoderma polysporum needs 15 - 20 days at 24°C on malt extract agar to form the conidiophores, which are both scattered but also in denser groups, above all on the edge of the culture vessel. The morphology of the bifruit form deviates in some features slightly from the description for this species (M.A. Rifai 1969: A REVISION OF THE GENUS TRICHODERMA in Mycological Papers, No. 116). Thus, the main branch of the conidia-bearers only reaches a transverse measurement of 2.0 - 3.8 ^a, while that for the species is indicated as 4 6.3 ^u. The phialides in the new strain of Trichoderma polysporum are, especially at the end of their development, elongated in length and this is typical for this species. Finally, the conidia are somewhat smaller than stated in the species description, namely 2.0 - 3.1 x 1.5 - 2.0 compared to 2.4 - 3.8 x 1.8 2.2^1. The present Trichoderma polysporum strain NRRL 8044 forms under all the mentioned cultivation conditions
ingen ekte klamydosporer.no true chlamydospores.
De nye soppstammer NRRL 5760 og NRRL 8044 lar seg dyrke på mange slags næringssubstrater, som inneholder de vanlige næringsstoffer for sopper. Således anvender disse stammer de for de karbon-heterotrofe organismer vanlig anvendte næringsstoffer, f.eks. sakkarose, glukose, maltose, laktose, stivelse, maltekstrakt som karbonkilde, organiske og uorganiske nitrogenholdige forbindelser, som maisstop, sojapepton, gjær- eller kj6ttekstrakter, natriumnitrat, ammoniumsulfat, ammoniumnitrat, aminosyrer etc, som nitrogenkilde såvel som de vanlige mineralsalter og spor-stoffer. The new mushroom strains NRRL 5760 and NRRL 8044 can be grown on many types of nutrient substrates, which contain the usual nutrients for mushrooms. Thus, these strains use the nutrients normally used for the carbon-heterotrophic organisms, e.g. sucrose, glucose, maltose, lactose, starch, malt extract as a carbon source, organic and inorganic nitrogen-containing compounds, such as corn starch, soy peptone, yeast or meat extracts, sodium nitrate, ammonium sulfate, ammonium nitrate, amino acids, etc., as a nitrogen source as well as the usual mineral salts and trace substances .
De nye antibiotika S 7481/F-l og S 7481/F-2 kan man fremstilleThe new antibiotics S 7481/F-1 and S 7481/F-2 can be produced
på den måte atin such a way that
1. Et flytende medium podes med en konidie- og mycel-suspensjon 1. A liquid medium is inoculated with a conidia and mycelium suspension
av stammen NRRL 5760 og kulturen inkuberes i 9 - 13 dager, foretrukket 11 dager, ved 24 - 30°C, foretrukket 2 7°C, og ved en pH verdi på 4,8 -8,8, foretrukket ved 8,1. Dyrkingen skjer i penicillinflasker under aerobe betingelser i en overflatekultur. of the strain NRRL 5760 and the culture is incubated for 9-13 days, preferably 11 days, at 24-30°C, preferably 27°C, and at a pH value of 4.8-8.8, preferably at 8.1. Cultivation takes place in penicillin bottles under aerobic conditions in a surface culture.
2. Et flytende medium podes med en konidie- og mycel-suspensjon 2. A liquid medium is inoculated with a conidia and mycelium suspension
av stammen NRRL 8044 og kulturen inkuberes i 11 - 18 dager, foretrukket 13 dager, ved 25 - 30°C, foretrukket ved 27°C, og ved en pH verdi på 4,3 - 6,2, foretrukket på 5,6 i et gjæringskar av stål under omroring (170 omdreininger pr. minutt) og lufting of the strain NRRL 8044 and the culture is incubated for 11 - 18 days, preferably 13 days, at 25 - 30°C, preferably at 27°C, and at a pH value of 4.3 - 6.2, preferably at 5.6 in a steel fermentation vessel under stirring (170 revolutions per minute) and aeration
(1 liter luft/minutt/liter næringslosning).(1 liter of air/minute/liter of nutrient solution).
Så snart en maksimal mengde antibiotika er produsert utvinnes disse fra dyrkingsbuljonen på i gg for seg kjent måte ved ekstraktive og/eller adsorptive metoder. As soon as a maximum quantity of antibiotics has been produced, these are extracted from the culture broth in a manner known per se by extractive and/or adsorptive methods.
En metode som har vist seg hensiktsmessig, består i ekstraksjon av det ved dyrkingen av soppstammen NRRL 5760 erholdte mycel, eventuelt etter foregående mekanisk oppdeling med 90% metanol, påfolgende avdamping av metanol og flere ganger ekstraksjon av den resulterende vandige blanding med etylenklorid. Til denne ekstrakt kan tilsettes den etylenkloridekstrakt som er utvunnet fra dyrkingsfiltratet. Det kan imidlertid også anvendes andre med vann ikke blandbare losningsmidler, f.eks. kloroform, etylacetat, metylenklorid eller butylacetat. A method that has proven to be appropriate consists in the extraction of the mycelium obtained from the cultivation of the mushroom strain NRRL 5760, optionally after previous mechanical division with 90% methanol, subsequent evaporation of methanol and extraction of the resulting aqueous mixture with ethylene chloride several times. To this extract can be added the ethylene chloride extract which is recovered from the cultivation filtrate. However, other water-immiscible solvents can also be used, e.g. chloroform, ethyl acetate, methylene chloride or butyl acetate.
Den organiske losningsinndampes til torrhet og resten avféttes enten ved kromatografering, f.eks. på kiselgel med kloroform med litt metanol, f.eks. i forholdet 98 : 2, eller ved fordeling meQilom petroleter og metanol, som er tilblandet en mengde vann tilstrekkelig for faseadskillelse. I det forste tilfelle forenes de mot Aspergillus niger antibiotisk virksomme fraksjoner, inndampes til torrhet og resten kromatograferes i metanolisk losning på "Sephadex LH 20". I det annet tilfelle konsentreres den metanolisk-vandige fase, eventuelt etter tilsetning av ytterligere vann, så langt at hovedmengden av metanolen er avdestillert, hvoretter den vandige blanding ekstraheres med etylenklorid, eller et annet av de ovennevnte, med vann ikke-blandbare losningsmidler, den resulterende organiske losninga inndampes til torrhet og resten kromatograferes i metanolisk losning på "Sephadex LH 20". Den videre oppdeling og rensing av de to antibiotika skjer ved fornyet kromatografering på aluminiumoksyd eller kiselgel, eller ved etterfSigende anvendelse av begge adsorpsjonsmidler. The organic solution is evaporated to dryness and the residue is defatted either by chromatography, e.g. on silica gel with chloroform with a little methanol, e.g. in the ratio 98:2, or by distribution with petroleum ether and methanol, which has been mixed with an amount of water sufficient for phase separation. In the first case, the fractions that are antibiotically active against Aspergillus niger are combined, evaporated to dryness and the residue chromatographed in methanolic solution on "Sephadex LH 20". In the second case, the methanolic-aqueous phase is concentrated, optionally after the addition of further water, until the main amount of the methanol has been distilled off, after which the aqueous mixture is extracted with ethylene chloride, or another of the above-mentioned, water-immiscible solvents, the the resulting organic solution is evaporated to dryness and the residue is chromatographed in methanolic solution on "Sephadex LH 20". The further separation and purification of the two antibiotics takes place by renewed chromatography on aluminum oxide or silica gel, or by subsequent application of both adsorbents.
En ytterligere metode, som har vist seg hensiktsmessig, består i ekstraksjon .av den ved dyrkingen av soppstammdn NRRL 8044 erholdte dyrkingsbuljong ved hjeip av et med vann ikkebblandbartlosnings-middel eventuelt etter foregående mekanisk oppdeling. Som løsningsmiddel kan fortrinnsvis anvendes etylenklorid, men kloroform, etylacetat, metylenklorid eller butylacetat kan også anvendes. Den organiske fase fraskilles, torres og inndampes. Resten kromatograferés, foretrukket med en metanol16sningjpå "Sephadex LH 20", idet de mot Aspergillus niger aktive fraksjoner forenes og kromatograferes en gang til på aluminiumoksyd med toluen som ble tilsatt 10 - 50% etylacetat. Påvisningen av de aktive substanser skjer også her ved aktivitetsprover overforAspergillus niger. Rensingen av de to antibiotika skjer ved A further method, which has proven to be appropriate, consists in the extraction of the culture broth obtained during the cultivation of mushroom strain NRRL 8044 with the help of a water-immiscible solvent, possibly after previous mechanical division. Ethylene chloride can preferably be used as a solvent, but chloroform, ethyl acetate, methylene chloride or butyl acetate can also be used. The organic phase is separated, dried and evaporated. The residue is chromatographed, preferably with a methanol solution on "Sephadex LH 20", as the fractions active against Aspergillus niger are combined and chromatographed once more on aluminum oxide with toluene to which 10 - 50% ethyl acetate has been added. The detection of the active substances also takes place here with activity samples against Aspergillus niger. The purification of the two antibiotics takes place by
fornyet kromatografering.renewed chromatography.
For fremstilling av de nye antibiotika lar seg også stammer anvende, som disse kan oppnås f.eks. ved seleksjon eller mutasjon under innvirkning av ultrafiolette eller rontgen-stråler eller ved anvendelse av andre forholdsregler, f.eks. ved behandling av laboratoriekulturer med egnede kjemikalier. For the production of the new antibiotics, strains can also be used, which can be obtained e.g. by selection or mutation under the influence of ultraviolet or X-rays or by the application of other precautions, e.g. when treating laboratory cultures with suitable chemicals.
De nye antibiotika S 7481/F-l og S 7481/F-2 utmerker seg ved interessante kjemoterapeutiske og farmakologiske egenskaper og kan folgelig anvendes som medisin. Således hemmer begge antibiotikaveksten avAspergillus niger. Spesielt utmerker substansen S 7481/F-l seg ved en immunosuppresiv virkning. Både S 7481/F-l og S 7481/F-2 viser dertil betennelseshemmende egenskaper. Det skal fremheves at begge antibiotika er lite sytostatisk aktive. The new antibiotics S 7481/F-1 and S 7481/F-2 are distinguished by interesting chemotherapeutic and pharmacological properties and can therefore be used as medicine. Thus, both antibiotics inhibit the growth of Aspergillus niger. In particular, the substance S 7481/F-1 is distinguished by an immunosuppressive effect. Both S 7481/F-1 and S 7481/F-2 also show anti-inflammatory properties. It should be emphasized that both antibiotics are not cytostatically active.
Den immunosuppresive virkning av S 7481/F-l kan vises påThe immunosuppressive effect of S 7481/F-1 can be shown on
folgende måte:following way:
a) Lokal hemolyse i gel in vitro, hjerne-proven:a) Local hemolysis in gel in vitro, the brain sample:
Hemming av hemolyse i % i forhold til kontrollprover utgjor vedInhibition of hemolysis in % compared to control samples constitutes wood
en dose på 2 x 60 mg/kg i.p. 99%, ved en dose på 1 x 150 mg/kg p.o. 90% og ved en dose på 3 x 200 mg/kg p.o. 99,5%. Både dannelsen av immunoglobulin M- og av immunoglobulin G-antistoffer hemmes. a dose of 2 x 60 mg/kg i.p. 99%, at a dose of 1 x 150 mg/kg p.o. 90% and at a dose of 3 x 200 mg/kg p.o. 99.5%. Both the formation of immunoglobulin M and immunoglobulin G antibodies is inhibited.
b) Hemagglutinasjonsprove (HAT) i mus:b) Hemagglutination test (HAT) in mice:
De mot-erutrocyter fra sau dannede antistoffer bestemmes. The anti-sheep erythrocyte antibodies are determined.
Hemmingen uttrykkes i suppresiy indeks (SI) idet den ubehandlende kontrollprove angis med 1,00. SI = 0,3 etter p.o. 5 x 200 mg/kg. The inhibition is expressed in suppression index (SI), with the non-treating control sample being indicated by 1.00. SI = 0.3 after p.o. 5 x 200 mg/kg.
c) Hud-transplantasjonsprove i mus:c) Skin transplantation sample in mice:
I H-2 histoin-tålende mus forlenges overlevelses-tidsforlengelsen for hudtransplantatet i forhold til ubehandlende kontrolldyr etter en dose på 10 x 200 mg/kg'p.o. i noen grad: ca. 16 dager lenger enn kontrollprovene. d) Ekspermimentell allergisk encefalomyelitis (EAE) i rotter: De eksperimentelt fremkalte nervevevs-skader, som lammet 8 av In H-2 histoin-tolerant mice, the survival time extension of the skin graft is prolonged compared to untreated control animals after a dose of 10 x 200 mg/kg'p.o. to some extent: approx. 16 days longer than the control samples. d) Experimental allergic encephalomyelitis (EAE) in rats: The experimentally induced nerve tissue damage, which paralyzed 8 of
10 dyr ved ubehandlende kontrolldyr, ga ved det samme antall 10 animals in untreated control animals, gave the same number
forsoksdyr som var blitt behandlet med 13 x 25 mg/kg i.p., en 100% beskyttelse. experimental animals that had been treated with 13 x 25 mg/kg i.p., a 100% protection.
e) Oksazolon-prove i mus:e) Oxazolone test in mice:
Avtagningen av oresvellingen uttrykkes som suppresiv indeks The decrease in ores swelling is expressed as suppressive index
(SI); SI = 0,6 etter 6 x 75 mg/kg p.o.(SAY); SI = 0.6 after 6 x 75 mg/kg p.o.
f) Zytopenier i knokemarg og blod:f) Cytopenia in bone marrow and blood:
Den hoye orale tilforsel av 6 x 500 mg/kg viser ingen tydelig The high oral intake of 6 x 500 mg/kg does not clearly show any
depresjon av de bloddannende celler i knokemargen og ingen leukopeni i blodet den 1, 3 og 7 dag etter den siste substans-tilforsel. depression of the blood-forming cells in the bone marrow and no leukopenia in the blood on the 1st, 3rd and 7th day after the last substance supply.
På grunn av deres immunosuppresive virkning kan forbindelsenDue to their immunosuppressive action, the compound can
S 7481/F-l anvendes for profylaks og behandling av sykdommer som anvendes i sammenheng med påvirkning av forsvarsreåksjonen på negativ måte. S 7481/F-l is used for the prophylaxis and treatment of diseases that are used in connection with influencing the defense reaction in a negative way.
De nye antibiotika S 7481/F-l S 7481/F-2 har likeledes en arthritishemmende virkning. Således virker de f.eks. i Freund-Adjuvans-Arhtritis-La.tenztidsforsok i rotter sterkt svellings-hemmende i doser på ca. 50 mg/kg kroppsvekt. The new antibiotics S 7481/F-1 S 7481/F-2 also have an anti-arthritis effect. Thus they work, e.g. in Freund-Adjuvans-Arhtritis-La.tenztidsorsok in rats strongly anti-swelling in doses of approx. 50 mg/kg body weight.
En tilsvarende virkning iakttas i Freund-Adjuvans-Arthritis-terapiforsok i rotter i doser på ca. 50 mg/kg/dag. A similar effect is observed in Freund-Adjuvant-Arthritis therapy trials in rats in doses of approx. 50 mg/kg/day.
På grunn av deres arthritishemmende virkning kan disse antibiotika anvendes for profylaks og behandling av arthritis og reumatiske sykdommer. Due to their anti-arthritis effect, these antibiotics can be used for the prophylaxis and treatment of arthritis and rheumatic diseases.
De doser som anvendes varierer selvf olgeli g alt etter typen av antibiotikum, tilforselsmåten og den tilstand som skal behandles. Vanlig oppnås dog i forsoksdyr tilfredsstillende resultater med en dose på 10 til 20 mg/kg kroppsvekt. Denne dose kan om nodvendig tilfores i 2 til 4deldoser eller også som retardform. For storre pattedyr ligger dågsdosen ved omtrent 50 til 900 mg. For oral tilforsel kan deldosene f.eks. inneholde omtrent 25 til 300 mg av de nye antibiotika ved siden av faste og flytende bærersubstanser. The doses used naturally vary according to the type of antibiotic, the method of administration and the condition to be treated. Usually, however, satisfactory results are achieved in experimental animals with a dose of 10 to 20 mg/kg body weight. If necessary, this dose can be given in 2 to 4 partial doses or also as a slow-release form. For larger mammals, the daily dose is approximately 50 to 900 mg. For oral administration, the partial doses can e.g. contain approximately 25 to 300 mg of the new antibiotics alongside solid and liquid carrier substances.
Som medisin kan de nye antibiotika tilfores alene eller i passende preparatform med farmakologisk indifferente hjelpestoffer. 1 de etterfølgende eksempler som skal illustrere oppfinnelsen nærmere, er alle temperaturangivelser i °C. As medicine, the new antibiotics can be administered alone or in suitable preparation form with pharmacologically indifferent excipients. 1 the following examples which will illustrate the invention in more detail, all temperature indications are in °C.
Eksempel 1.Example 1.
oo
10 liter næringsløsning som pr. liter inneholder 30 g sakkarose, 10 gram mais-stop, 3 gram NaNO^, 1 gram K^HPC^, 0,5 g MgS04 x 7H20, 0,5 gram KC1 og 0,01 gram FeS04 x 7H20 podes med .100 ml av en konidie- og mycel-suspensjon av stammen NRRL 5760 og inkuberes i penicillinflasker med hver 700 ml rominnhold i 11 dager ved 27°C. 10 liters of nutrient solution which per liter contains 30 g sucrose, 10 grams corn-stop, 3 grams NaNO^, 1 gram K^HPC^, 0.5 g MgS04 x 7H20, 0.5 gram KC1 and 0.01 gram FeS04 x 7H20 inoculated with .100 ml of a conidial and mycelial suspension of the strain NRRL 5760 and incubated in penicillin bottles of 700 ml volume each for 11 days at 27°C.
Det fra dyrkingsbuljongen fraskilte mycel ekstraheres ved knusing og omroring med 3,5 liter 90% metanol og det ved avsuging på filter fra losningsmidlet adskilte, knuste mycel behandles ytterligere 2 ganger på den samme måte med 90% metanol. De forenede filtrater inndampes i vakuum ved 40°C.badtemperatur så langt at væsken hovedsakelig bare består av vann. Den resulterende blanding ekstraheres 6 ganger med det samme volum etylenklorid ved utristing, hvoretter de forenede etylenklorid-losninger renses ved utrysting med vann og inndampes i vakuum ved 40°C badtemperatur. Den således erholdte rest kromatograferes på 2 50 gram kiselgel (kiselgel 60 "Merck", kornstorrelse 0,063 - 0,200 mm) under anvendelse av kloroform med en tilsetning av 2% metanol som elueringsvæske, som oppfanges i 200 ml fraksjoner. De ved plate-diffusjonstest mot Aspergillus niger antibiotisk virksomme fraksjoner forenes, inndampes til torrhet på beskrevet måte og kromotograferes etter losning i metanol på ilO gram "Sephadex LH 20" med det samme løsningsmiddel, hvoretter man forener de 20 ml fraksjoner som ved den samme prove som ovenfor har vist seg antibiotisk virksom mot Aspergillus niger. Ved undersøkelsen i tynnsjikt kromatogram, f.eks. ved hjelp av kiselgel på "Polygram"-folier og heksan-aceton (1:1) som flytemiddel viser det seg at resten av den på den ovenfor beskrevne måte inndampede metanollosning hovedsakelig består av de to nye substanser S 7481/F-l og S 7481/F-2. De skilles og renses samtidig, idet deres blanding kromatograferes på nytt under anvendelse av den tusendobbelte mengde kiselgel av den ovennevnte, kvalitet og kloroform som er tilsatt 2% metanol. Undérsokelsen av eluatfraksjonene, hvis volum i ml er halvparten så stort som vekten av kiselgel i gram, ved tynnsjiktkromatogram viser at substansen S 7481/F-l forst kommer til syne i eluatet fulgt av en blanding av de to substanser og deretter av enhetlig S 7481/F-2. Fra blandingen kan under de samme betingelser ved gjentatt kromatografering utvinnes ytterligere mengder av de to substanser. The mycelium separated from the culture broth is extracted by crushing and stirring with 3.5 liters of 90% methanol and the crushed mycelium separated by suction on a filter from the solvent is treated a further 2 times in the same way with 90% methanol. The combined filtrates are evaporated in a vacuum at 40° C. bath temperature to such an extent that the liquid mainly consists only of water. The resulting mixture is extracted 6 times with the same volume of ethylene chloride by shaking, after which the combined ethylene chloride solutions are purified by shaking with water and evaporated in vacuo at 40°C bath temperature. The residue thus obtained is chromatographed on 250 grams of silica gel (silica gel 60 "Merck", grain size 0.063 - 0.200 mm) using chloroform with an addition of 2% methanol as eluent, which is collected in 200 ml fractions. The fractions that are antibiotically active in the plate diffusion test against Aspergillus niger are combined, evaporated to dryness in the described manner and chromatographed after dissolving 10 grams of "Sephadex LH 20" in methanol with the same solvent, after which the 20 ml fractions as for the same sample are combined as above has been shown to be antibiotically effective against Aspergillus niger. In the examination in thin-layer chromatogram, e.g. with the help of silica gel on "Polygram" foils and hexane-acetone (1:1) as a fluid, it turns out that the rest of the methanol solution evaporated in the manner described above mainly consists of the two new substances S 7481/F-l and S 7481/ F-2. They are separated and purified at the same time, their mixture being chromatographed again using a thousandfold amount of silica gel of the above quality and chloroform to which 2% methanol has been added. The examination of the eluate fractions, whose volume in ml is half as large as the weight of silica gel in grams, by thin layer chromatogram shows that the substance S 7481/F-l first appears in the eluate followed by a mixture of the two substances and then by uniform S 7481/F -2. Further quantities of the two substances can be recovered from the mixture under the same conditions by repeated chromatography.
Det nye antibiotikum S 7481/F-l har fSigende egenskaper:The new antibiotic S 7481/F-l has the following properties:
Amorft, fargelSst pulver med smeltepunkt 137 - 140°C (spalting); Amorphous, colorless powder with melting point 137 - 140°C (decomposition);
/ a7p° -236° (CHC13, C 0,5) / a7p° -236° (CHC13, C 0.5)
UV: se fig. 1.UV: see fig. 1.
IR: se fig. 2.IR: see fig. 2.
Proton-NMR: se fig. 3.Proton NMR: see fig. 3.
13 13
C-NMR: se tabellen.C-NMR: see the table.
Massepektrum (CEC massespektrometer 21-1108, ionekilde E.B.: InnfSringssystem:direkte, elektronenergi: 70 eV, ioneaksellerasjon: 4 kV, temperatur for ionekilden, 290°C, trykk: 5.IO<-6>Torr, Mass spectrum (CEC mass spectrometer 21-1108, ion source E.B.: Introduction system: direct, electron energy: 70 eV, ion acceleration: 4 kV, temperature of the ion source, 290°C, pressure: 5.IO<-6>Torr,
Peak-Matching-fremgangsmåten, refereransesubstans "PCR 8" /Tris-pentadekafluorheptyl-1,3,5-triazin, M=1184, 9374/: hSyeste massetopp m/e = 1183,831 (+ 0,004), tilsvarende massen av ionet C62<H>109N11<<>^11' Pro<^u^tet fra eliminering av vann fra det virkelig molekyl C62<H>iiiNn<0>i2<*>LSselighet: lettlSselig i de fleste vanlige organiske ISsningsmidler, praktisk ulSselig i petroleter og vann. Opptreden i tynnsjikt-kromatogram (substansflekker gjort Peak-Matching procedure, reference substance "PCR 8" /Tris-pentadecafluoroheptyl-1,3,5-triazine, M=1184, 9374/: hSyest mass peak m/e = 1183.831 (+ 0.004), corresponding to the mass of the ion C62 <H>109N11<<>^11' Pro<^u^tet from the elimination of water from the real molecule C62<H>iiiNn<0>i2<*>LSolubility: easily soluble in most common organic solvents, practically insoluble in petroleum ether and water. Appearance in thin-layer chromatogram (substance stains made
-synlige med joddamp):-visible with iodine vapor):
a) Kiselgel på glassplater: kloroform + 4% metanol som flytemiddel: Rf = 0,52. b) Kiselgel på folier " polygram"; heksan/aceton 1 : 1 som flytemiddel: Rf = 0,37; kloroform/aceton 2 : 1 som flytemiddel: Rf = 0,34. a) Silica gel on glass plates: chloroform + 4% methanol as emulsifier: Rf = 0.52. b) Silica gel on foils "polygram"; hexane/acetone 1 : 1 as eluent: Rf = 0.37; chloroform/acetone 2 : 1 as eluent: Rf = 0.34.
Elementæranalyse (proven lost i benzen, losningen inndampet til torrhet, resten torret i hoyvakuum i 5 timer ved 100°C): funnet C 61,8 H 9,4 N 13,0 O 15,7%. Elemental analysis (the sample dissolved in benzene, the solution evaporated to dryness, the residue dried in high vacuum for 5 hours at 100°C): found C 61.8 H 9.4 N 13.0 O 15.7%.
De verdier som ble funnet stemmer overens med bruttoformlen, The values found agree with the gross formula,
<C>62<H>111<N>11°12- <C>62<H>111<N>11°12-
I hydrolysatet, som erholdes ved koking av forbindelsen S 7481/F-l med 6 N saltsyre (20 timer), kan aminosyren alanin, a-amino-smorsyre, N-metylleucin, N-metylglycin og valin påvises. Det In the hydrolyzate, which is obtained by boiling the compound S 7481/F-1 with 6 N hydrochloric acid (20 hours), the amino acid alanine, α-aminobutyric acid, N-methylleucine, N-methylglycine and valine can be detected. The
nye antibiotikum S 7481/F-2 har fSigende egenskaper:new antibiotic S 7481/F-2 has the following properties:
Amorft, fargelSst pulver med smeltepunkt 127 - 130°C ;(;spalting) • /o/£° = -243° (kloroform, c = 0,5) Amorphous, colorless powder with melting point 127 - 130°C ;(;cleavage) • /o/£° = -243° (chloroform, c = 0.5)
UV: se fig. 4.UV: see fig. 4.
IR: se fig. 5.IR: see fig. 5.
Proton-NMR: se fig. 6.Proton NMR: see fig. 6.
Massespektrum (opptagningsbetingelser se antibiotikum S 7481/F-l): HSyeste massetopp m/e = 1169,815 (+ 0,006), tilsvarende massen av ionet C5i<H>io7Nll°ll'Proc^u^tet fra eliminering av vann fra molekylet<Gg>1<H>l09<N>ii<o>i2<*>Mass spectrum (recording conditions see antibiotic S 7481/F-l): HSyest mass peak m/e = 1169.815 (+ 0.006), corresponding to the mass of the ion C5i<H>io7Nll°ll'Proc^u^tet from the elimination of water from the molecule<Gg >1<H>l09<N>ii<o>i2<*>
Loselighet: lettloselig i de fleste vanlige organiske losningsmidler-: praktisk uloselig i petroleter og vann. Solubility: easily soluble in most common organic solvents-: practically insoluble in petroleum ether and water.
Forhold i tynnsjiktkromotogram (substansflekker gjort synlige med j oddamp): a) Kiselgel på glassplater; kloroform + 4% metanol som flytemiddel: Rf =. 0,46. Conditions in thin-layer chromatogram (substance spots made visible with j odd vapour): a) Silica gel on glass plates; chloroform + 4% methanol as eluent: Rf =. 0.46.
b) .Kiselgel på folier "polygram"; heksan/aceton 1 : 1 som flytemiddel: Rf = 0,28; kloroform/aceton 2 : 1 som flyte- b) .Silica gel on foils "polygram"; hexane/acetone 1 : 1 as eluent: Rf = 0.28; chloroform/acetone 2 : 1 as liquid
middel: Rf = 0,26.mean: Rf = 0.26.
Elementæranalyse (prove torret i 5 timer i hoyvakuum ved 100°C): Funnet C 61,7 H 9,1 N 13,1 O 16,5%. Elemental analysis (sample dried for 5 hours in high vacuum at 100°C): Found C 61.7 H 9.1 N 13.1 O 16.5%.
De verdier som ble funnet stemmer overens med bruttoformlen<C>61<H>109<N>11°12' The values found agree with the gross formula<C>61<H>109<N>11°12'
Den som utgangsmaterial for podingen anvendte konidie- og mycel-suspensjon fremstilles fra en kultur av den opprinnelig isolerte stamme NRRL 5760, som ble dyrket i 10 dager på et agarmedium, som pr. liter inneholdt 20 gram maltekstrakt, 20 agar, 4 gram gjæreks trakt og avmineralisert vann. Konidiene og mycelet ble opptatt i fysiologisk koksaltlosning. Denne suspensjon tjener til poding av den ovennevnte næringsløsning. The conidia and mycelium suspension used as starting material for the inoculation is prepared from a culture of the originally isolated strain NRRL 5760, which was grown for 10 days on an agar medium, which per liter contained 20 grams of malt extract, 20 grams of agar, 4 grams of garlic extract and demineralized water. The conidia and mycelium were absorbed in physiological saline solution. This suspension serves for inoculation of the above nutrient solution.
Eksempel 2:Example 2:
2 50 liter av en næringsløsning som pr. liter inneholdt 30 gram sakkarose, 10 gram mais=stop, 3 gram NaNO^/1 gram K2HP04, 0,5 2 50 liters of a nutrient solution that per liter contained 30 grams sucrose, 10 grams corn=stop, 3 grams NaNO^/1 gram K2HP04, 0.5
gram MgS04x 7H20, 0,5 gram KC1 og 0,01 gram FeSC>4x 7H20 ble podet med 2,5 liter, konidie- og mycel-suspensjon av stammen NRRL 5760 og inkubert i penicillinflasker med hver 700 ml rominnhold i 11 dager ved 27°C. gram MgS04x 7H2O, 0.5 gram KC1 and 0.01 gram FeSC>4x 7H2O were inoculated with 2.5 liters, conidial and mycelial suspension of strain NRRL 5760 and incubated in penicillin bottles of each 700 ml room content for 11 days at 27 °C.
Frå dyrkingsbuljongen fraskilles mycelet ved sentrifugering og homogeniseres i en ultra-hurtigmikser med 80 liter 90% metanol, deretter skilles den metanoliske ekstraktlosning ved sentrifugering From the culture broth, the mycelium is separated by centrifugation and homogenized in an ultra-fast mixer with 80 liters of 90% methanol, then the methanolic extract solution is separated by centrifugation
-fra faststoffet og det siste ekstraheres enda to ganger på den samme måte med 90% metanol. De forenede ekstraktlosninger konsentreres i vakuum ved 30 - 40°C til 15 liter. Den tilbake-blivende vandige fase ekstraheres 8 ganger med det samme volum-etylenklorid. Hver av etylenkloridekstraktene vaskes med 5 liter vann. Etylenkloridlosningene inndampes etter forening i vakuum ved 30 - 40°C. For avfetting fordeles ekstraktresten på den måte mellom petroleter og 90% metanol at den etter losning i 2 liter 90% metanol i rekkefolge rystes 3 ganger med hver gang 2 liter petroleter (kokepunkt 30 - 35°C), som befinner seg i 3 ryste-trakter, hvoretter de 3 petroleterfaser etter hverandre enda, ekstraheres 2 ganger med hver gang 2 liter 90% metanol. Til de forenede metanoliske losninger tilsettes 3 liter vann og blandingen konsentres i vakuum ved 30 - 40°C til et volum på 2 liter. Det vandige konsentrat ekstraheres ved 5 gangers utrysting med hver gang 2 liter etylenklorid, etylenkloridlosningene vaskes med hver gang 0,5 liter vann, forenes og inndampes til torrhet i vakuum ved 30 - 40°C.. Resten underkastes kromatografering på den tjuedobbelte mengde "Sephadex LH 20 ", hvorved metanol anvendes som elueringsmiddel. Eluatet oppfanges i 20 ml fraksjoner og de av fraksjonene som viser seg antibiotisk virksomme mot Aspergillus niger forenes og inndampes i vakuum ved 30 - 40°C -from the solid and the latter is extracted two more times in the same way with 90% methanol. The combined extract solutions are concentrated in vacuo at 30 - 40°C to 15 litres. The remaining aqueous phase is extracted 8 times with the same volume of ethylene chloride. Each of the ethylene chloride extracts is washed with 5 liters of water. The ethylene chloride solutions are evaporated after union in vacuum at 30 - 40°C. For degreasing, the extract residue is distributed between petroleum ether and 90% methanol in such a way that, after dissolving in 2 liters of 90% methanol, it is successively shaken 3 times with each time 2 liters of petroleum ether (boiling point 30 - 35°C), which is in 3 shaking funnels, after which the 3 petroleum ether phases, one after the other, are extracted 2 times with 2 liters of 90% methanol each time. 3 liters of water are added to the combined methanolic solutions and the mixture is concentrated in vacuo at 30 - 40°C to a volume of 2 litres. The aqueous concentrate is extracted by shaking out 5 times with 2 liters of ethylene chloride each time, the ethylene chloride solutions are washed with 0.5 liters of water each time, combined and evaporated to dryness in vacuum at 30 - 40°C. The residue is subjected to chromatography on a twenty-fold amount of "Sephadex LH 20", whereby methanol is used as eluent. The eluate is collected in 20 ml fractions and those of the fractions that prove antibiotically active against Aspergillus niger are combined and evaporated in a vacuum at 30 - 40°C
til torrhet. Deretter kromatograferes resten på den syttidobbelte mengde aluminiumoksyd (noytral) med aktivitetsgrad 1, hvorved blandinger av toluen og etylacetat tjener for eluering. Med toluen som er tilsatt 15% etyleacetat kommer i de forste 10 fraksjoner (volumet av fraksjonene: halvparten så mange liter som adsorpsjonsmiddel i kg) forst hovedmengden av substansen S 7481/F-l til syne, fulgt av en blanding av S 7481/F-l og 7481/F-2.. En ytterligere mengde av den samme blanding isoleres sammen med litt fremmed-substans ved etterfolgende eluering med toluen-etylacetat (1 : 1). Påvisningen av substansene i eluatfraksjonene skjer på tynnsjikt-kromatograf isk måte, f .eks.? ved hjelp av kiselgel på "polygram"-folier og heksanaceton 1:1) som flytemiddel. Fra de forenede to dryness. The residue is then chromatographed on seventy-fold the amount of aluminum oxide (neutral) with activity level 1, whereby mixtures of toluene and ethyl acetate are used for elution. With toluene to which 15% ethyl acetate has been added, in the first 10 fractions (the volume of the fractions: half as many liters as the adsorbent in kg) first the main amount of the substance S 7481/F-l appears, followed by a mixture of S 7481/F-l and 7481 /F-2.. A further amount of the same mixture is isolated together with a little extraneous substance by subsequent elution with toluene-ethyl acetate (1:1). The detection of the substances in the eluate fractions takes place by thin-layer chromatography, e.g.? using silica gel on "polygram" foils and hexaneacetone 1:1) as a fluid. From the united
fraksjoner, som inneholder substansen S 7481/F-l i enhetlig form, utvinnes substansen ved inndamping i vakuum ved 30 - 40°C og etterfSigende tSrring av resten i hSyvakuum ved 50°C. De forenede eluatfraksjoner, som inneholder blandingen, inndampes likeledes under de samme betingelser. For isolering av ytterligere S 7481/F-l og av enhetlig S 7481/F-2 underkastes resten kromatografering på den tusendobbelte mengde kiselgel, idet kloroform, som innehol<*>der 2% metanol, anvendes som elueringsmiddel som dette og de videre foranstaltniger ble beskrevet i eksempel 1-fractions, which contain the substance S 7481/F-1 in uniform form, the substance is recovered by evaporation in vacuum at 30 - 40°C and subsequent drying of the residue in high vacuum at 50°C. The combined eluate fractions, which contain the mixture, are likewise evaporated under the same conditions. For the isolation of additional S 7481/F-1 and of uniform S 7481/F-2, the residue is subjected to chromatography on a thousand-fold amount of silica gel, with chloroform, which contains 2% methanol, being used as eluent as this and the further measures were described in example 1-
Eksempel 3.Example 3.
500 liter av en næringslSsning, som pr. liter inneholdt 40 g glukose, 5 gram kaseinpepton, 5 g MgS04xWH^ O, 2 gram KH2PC>4500 liters of a nutrient solution, which per liter contained 40 g glucose, 5 grams casein peptone, 5 g MgS04xWH^ O, 2 grams KH2PC>4
3 gram NaNO^-, 0,5 gram KC1, 0,01 gram FeS04og avmineralisert vann, ble podet med 50 liter av en forkultur av stammen NRRL 3 grams of NaNO^-, 0.5 grams of KC1, 0.01 grams of FeSO4 and demineralized water, was inoculated with 50 liters of a pre-culture of the strain NRRL
8044 og inkubert i et gjæringskar av stål under omrSring (170 omdreininger pr. minutt) og lufting 01 liter luft/minutt/liter næringslSsning) i 13 dager ved 27°C. 8044 and incubated in a steel fermentation vessel under rotation (170 revolutions per minute) and aeration (01 liter of air/minute/liter of nutrient solution) for 13 days at 27°C.
500 liter dyrkingsbuljong knuses i ultra-hurtigmixer og ekstraheres flere ganger med hver gang 500 liter 1,2-dikloretan. Den organiske fase fraskilles, tSrres over natriumsulfat og inndampes i vakuum. Inndampningsresten kromatograferes på den hundredobbelte mengde "Sephadex LH 20" med metanol. De mot-Aspergillus niger aktive fraksjoner forenes og blandingen kromatograferes pa den hundre til tohundre dobbelte mengde nSytralt aluminiumoksyd (aktivitetsgrad I). Elueringen foregår med toluen som var tilsatt 10 - 50% etylacetat. Det elueres fSrst overveiende biprodukter, deretter S 7481/F-l og deretter S 7481/F-2. Påvisningen av de aktive stoffer i fraksjonene skjer også her ved aktivitetsprSving overfor Aspergillus niger. For renhetskontroll foretas tynnsjiktkromatografisk undersSkelse på kiselgelplater med kloroformmetanol (96 : 4), idet antibiotika-forbindelsene gjSres synlige med joddamp. De rene fraksjoner forenes og utgnis med heksan, idet antibiotikaforbindelse med S 7481/F-l og S 7481/F-2 erholdes i form av et hvitt amorft 500 liters of culture broth are crushed in an ultra-fast mixer and extracted several times with 500 liters of 1,2-dichloroethane each time. The organic phase is separated, concentrated over sodium sulphate and evaporated in vacuo. The evaporation residue is chromatographed on a hundredfold amount of "Sephadex LH 20" with methanol. The anti-Aspergillus niger active fractions are combined and the mixture is chromatographed on the one hundred to two hundred double quantity of nCytral aluminum oxide (activity level I). The elution takes place with toluene to which 10 - 50% ethyl acetate has been added. Predominantly by-products are eluted first, then S 7481/F-1 and then S 7481/F-2. The detection of the active substances in the fractions also takes place here by activity testing against Aspergillus niger. For purity control, thin-layer chromatographic analysis is carried out on silica gel plates with chloroform-methanol (96:4), the antibiotic compounds being made visible with iodine vapour. The pure fractions are combined and triturated with hexane, the antibiotic compound with S 7481/F-1 and S 7481/F-2 being obtained in the form of a white amorphous
o o
pulver om torres i hoyvakuum ved 50°C.powder if dried in high vacuum at 50°C.
Den som utgangsmaterial anvendte forkultur erholdes på folgende måte: Den for poding anvendte spore- og mycelsuspensjon fremstilles fra en kultur av den opprinnelig isolerte stamme NRRL 8044, som var dyrket i 21 dager ved 27°C på et agarmedium som pr. liter inneholdt 20 gram maltekstrakt, 20 agar, 4 gram gjærekstrakt og avmineralisert vann. Sporene og mycelet i denne kultur ble opptatt i fysilogisk koksaltlosning. The pre-culture used as starting material is obtained in the following way: The spore and mycelial suspension used for inoculation is prepared from a culture of the originally isolated strain NRRL 8044, which was grown for 21 days at 27°C on an agar medium which per liter contained 20 grams of malt extract, 20 grams of agar, 4 grams of yeast extract and demineralized water. The spores and mycelium in this culture were immersed in physiological saline solution.
Med denne suspensjon ble 50 liter av en næringslSsning, som With this suspension, 50 liters of a nutrient solution, which
hadde den samme sammensetning som i det 500 liter's gjæringskar av stål, podet og inkubert i et gjæringskar av stål under omrSring (200 -omdreininger pr. minutt) og lufting (1 liter luft/ minutt/liter næringslSsning) i 3 dager ved 27°C. Denne gjærings-lSsning tjener som podematerial for det 500 1iter"s gjæringskar av stål. had the same composition as in the 500 liter steel fermenter, inoculated and incubated in a steel fermenter under agitation (200 revolutions per minute) and aeration (1 liter of air/minute/liter of nutrient solution) for 3 days at 27°C . This fermentation solution serves as seed material for the 500 liter steel fermentation vessel.
Claims (3)
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CH1711173A CH589716A5 (en) | 1973-12-06 | 1973-12-06 | Antibiotics S 7481-F-1 and 2 - prepd. from new strains of Cylindrocarbon or Trichoderma esp. useful as immunosuppressant |
| CH1404374A CH603790A5 (en) | 1974-10-21 | 1974-10-21 | Antibiotics S 7481-F-1 and 2 |
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| Publication Number | Publication Date |
|---|---|
| NO744292L true NO744292L (en) | 1975-06-30 |
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| NO744292A NO744292L (en) | 1973-12-06 | 1974-11-28 |
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| DE (2) | DE2455859C2 (en) |
| DK (1) | DK136780B (en) |
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| FI (2) | FI52851C (en) |
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| CH614931A5 (en) | 1975-11-04 | 1979-12-28 | Sandoz Ag | |
| US4117118A (en) * | 1976-04-09 | 1978-09-26 | Sandoz Ltd. | Organic compounds |
| DE2907460A1 (en) * | 1978-03-07 | 1979-09-13 | Sandoz Ag | NEW RESORBABLE GALENIC COMPOSITIONS |
| SE448386B (en) * | 1978-10-18 | 1987-02-16 | Sandoz Ag | NEW CYCLOSPORIN DERIVATIVES, PROCEDURE FOR PREPARING THEM AND PHARMACEUTICAL COMPOSITION CONTAINING THEM |
| EP0098457B1 (en) * | 1980-02-14 | 1987-08-05 | Sandoz Ag | (1s,2r,3r)- and (1r,2s,3s)-nitrilo-1-carbonyl-3-methyl-2-oxy-heptanes and -hept-5-enes useful in the total synthesis of cyclosporins and processes for their production |
| US4396542A (en) | 1980-02-14 | 1983-08-02 | Sandoz Ltd. | Method for the total synthesis of cyclosporins, novel cyclosporins and novel intermediates and methods for their production |
| EP0056782B1 (en) | 1981-01-09 | 1984-08-01 | Sandoz Ag | Novel cyclosporins |
| JPS59129920U (en) * | 1983-02-16 | 1984-08-31 | 日立造船株式会社 | Opening/closing device for bypass valve with gate |
| GB8717300D0 (en) * | 1987-07-22 | 1987-08-26 | Nat Res Dev | Cyclosporins |
| ES2095926T5 (en) | 1990-11-02 | 2001-02-16 | Novartis Ag | CYCLOSPORINS |
| DE69222150T2 (en) * | 1991-01-25 | 1998-01-15 | Fujisawa Pharmaceutical Co | PROCESS FOR THE PRODUCTION OF CYCLOSPORIN-A AND / OR C |
| KR100304324B1 (en) * | 1994-10-13 | 2001-11-22 | 김용규 | Method for manufacturing cyclosporine a |
| HUT77283A (en) | 1994-11-03 | 1998-03-30 | Novartis Ag. | Novel cyclosporine preparation forms for oral administration of simple composition and high bio-availability, and process for producing them |
| US6423233B1 (en) | 2000-08-15 | 2002-07-23 | Biogal Gyogyszergyar Rt. | Purification process |
| CA2518265A1 (en) | 2003-03-17 | 2004-09-30 | Albany Molecular Research, Inc. | Novel cyclosporins |
| US7511013B2 (en) | 2004-09-29 | 2009-03-31 | Amr Technology, Inc. | Cyclosporin analogues and their pharmaceutical uses |
| US7378391B2 (en) | 2004-09-29 | 2008-05-27 | Amr Technology, Inc. | Cyclosporin alkyne analogues and their pharmaceutical uses |
| US7361636B2 (en) | 2004-10-06 | 2008-04-22 | Amr Technology, Inc. | Cyclosporin alkynes and their utility as pharmaceutical agents |
| US7696165B2 (en) | 2006-03-28 | 2010-04-13 | Albany Molecular Research, Inc. | Use of cyclosporin alkyne analogues for preventing or treating viral-induced disorders |
| US7696166B2 (en) | 2006-03-28 | 2010-04-13 | Albany Molecular Research, Inc. | Use of cyclosporin alkyne/alkene analogues for preventing or treating viral-induced disorders |
| EP2151450A1 (en) * | 2008-07-29 | 2010-02-10 | Sandoz AG | Method for processing microbiologically produced cyclic oligopeptides |
-
1974
- 1974-11-26 DE DE2455859A patent/DE2455859C2/en not_active Expired
- 1974-11-26 DE DE2463138A patent/DE2463138C2/en not_active Expired
- 1974-11-27 FI FI743434A patent/FI52851C/en active
- 1974-11-27 DK DK617074AA patent/DK136780B/en unknown
- 1974-11-28 NO NO744292A patent/NO744292L/no unknown
- 1974-11-29 SE SE7415027A patent/SE423720B/en not_active IP Right Cessation
- 1974-12-02 NL NLAANVRAGE7415682,A patent/NL179220C/en not_active IP Right Cessation
- 1974-12-02 GB GB52013/74A patent/GB1491509A/en not_active Expired
- 1974-12-04 IL IL46180A patent/IL46180A/en unknown
- 1974-12-04 AU AU76081/74A patent/AU500889B2/en not_active Expired
- 1974-12-04 DD DD182775A patent/DD115695A5/xx unknown
- 1974-12-04 IE IE2502/74A patent/IE40549B1/en not_active IP Right Cessation
- 1974-12-04 NZ NZ176117A patent/NZ176117A/en unknown
- 1974-12-04 ES ES432559A patent/ES432559A1/en not_active Expired
- 1974-12-05 FR FR7439877A patent/FR2253531B1/fr not_active Expired
- 1974-12-05 JP JP13893874A patent/JPS5615235B2/ja not_active Expired
- 1974-12-05 CA CA215,287A patent/CA1030469A/en not_active Expired
-
1977
- 1977-04-15 FI FI771201A patent/FI54606C/en not_active IP Right Cessation
- 1977-04-22 PH PH19702A patent/PH13773A/en unknown
-
1980
- 1980-09-25 HK HK538/80A patent/HK53880A/en unknown
-
1981
- 1981-12-30 MY MY203/81A patent/MY8100203A/en unknown
Also Published As
| Publication number | Publication date |
|---|---|
| CA1030469A (en) | 1978-05-02 |
| MY8100203A (en) | 1981-12-31 |
| NZ176117A (en) | 1981-05-01 |
| NL179220B (en) | 1986-03-03 |
| DE2455859A1 (en) | 1975-06-12 |
| DK136780C (en) | 1978-05-01 |
| DE2463138C2 (en) | 1984-07-05 |
| FR2253531A1 (en) | 1975-07-04 |
| IL46180A (en) | 1978-07-31 |
| DK617074A (en) | 1975-07-28 |
| ES432559A1 (en) | 1977-03-01 |
| NL179220C (en) | 1986-08-01 |
| FI343474A (en) | 1975-06-07 |
| JPS5615235B2 (en) | 1981-04-09 |
| DE2455859C2 (en) | 1983-12-15 |
| IE40549B1 (en) | 1979-06-20 |
| FI52851B (en) | 1977-08-31 |
| FI54606C (en) | 1979-01-10 |
| FI52851C (en) | 1977-12-12 |
| NL7415682A (en) | 1975-06-10 |
| AU500889B2 (en) | 1979-06-07 |
| IL46180A0 (en) | 1975-03-13 |
| IE40549L (en) | 1975-06-06 |
| HK53880A (en) | 1980-10-03 |
| JPS5089598A (en) | 1975-07-18 |
| FI771201A (en) | 1977-04-15 |
| GB1491509A (en) | 1977-11-09 |
| AU7608174A (en) | 1976-06-10 |
| DK136780B (en) | 1977-11-21 |
| PH13773A (en) | 1980-09-23 |
| SE423720B (en) | 1982-05-24 |
| FR2253531B1 (en) | 1978-07-21 |
| SE7415027L (en) | 1975-06-09 |
| FI54606B (en) | 1978-09-29 |
| DD115695A5 (en) | 1975-10-12 |
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