CN108992437A - Purposes of the lauroyl arginine ethyl ester as antibacterial agent for animals - Google Patents
Purposes of the lauroyl arginine ethyl ester as antibacterial agent for animals Download PDFInfo
- Publication number
- CN108992437A CN108992437A CN201810649050.0A CN201810649050A CN108992437A CN 108992437 A CN108992437 A CN 108992437A CN 201810649050 A CN201810649050 A CN 201810649050A CN 108992437 A CN108992437 A CN 108992437A
- Authority
- CN
- China
- Prior art keywords
- lae
- compound
- medicament
- formula
- administered
- 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.)
- Granted
Links
- 241001465754 Metazoa Species 0.000 title claims abstract description 79
- XTJKNGLLPGBHHO-HNNXBMFYSA-N (2s)-5-(diaminomethylideneamino)-2-(dodecanoylamino)pentanoic acid Chemical compound CCCCCCCCCCCC(=O)N[C@H](C(O)=O)CCCN=C(N)N XTJKNGLLPGBHHO-HNNXBMFYSA-N 0.000 title claims abstract description 67
- 239000003242 anti bacterial agent Substances 0.000 title abstract description 37
- 241000894006 Bacteria Species 0.000 claims abstract description 83
- 241000588724 Escherichia coli Species 0.000 claims abstract description 49
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 48
- 201000010099 disease Diseases 0.000 claims abstract description 45
- 241001478212 Riemerella anatipestifer Species 0.000 claims abstract description 38
- 244000144972 livestock Species 0.000 claims abstract description 37
- 241000191967 Staphylococcus aureus Species 0.000 claims abstract description 32
- 230000001717 pathogenic effect Effects 0.000 claims abstract description 26
- 244000144977 poultry Species 0.000 claims abstract description 23
- 208000035143 Bacterial infection Diseases 0.000 claims abstract description 20
- 208000015181 infectious disease Diseases 0.000 claims abstract description 18
- 241000251468 Actinopterygii Species 0.000 claims abstract description 9
- 208000022362 bacterial infectious disease Diseases 0.000 claims abstract description 8
- 239000003814 drug Substances 0.000 claims description 101
- 150000001875 compounds Chemical class 0.000 claims description 92
- 230000037396 body weight Effects 0.000 claims description 35
- 239000000243 solution Substances 0.000 claims description 28
- 241000272525 Anas platyrhynchos Species 0.000 claims description 22
- 210000000170 cell membrane Anatomy 0.000 claims description 22
- 125000004432 carbon atom Chemical group C* 0.000 claims description 14
- 206010012735 Diarrhoea Diseases 0.000 claims description 13
- 238000004519 manufacturing process Methods 0.000 claims description 11
- 244000000010 microbial pathogen Species 0.000 claims description 10
- 244000005700 microbiome Species 0.000 claims description 10
- 241000589562 Brucella Species 0.000 claims description 8
- 241000287828 Gallus gallus Species 0.000 claims description 8
- 241000606790 Haemophilus Species 0.000 claims description 8
- 241000606860 Pasteurella Species 0.000 claims description 8
- 241000607142 Salmonella Species 0.000 claims description 8
- 102000003777 Interleukin-1 beta Human genes 0.000 claims description 7
- 108090000193 Interleukin-1 beta Proteins 0.000 claims description 7
- 230000002265 prevention Effects 0.000 claims description 7
- 150000003839 salts Chemical class 0.000 claims description 7
- 241000193738 Bacillus anthracis Species 0.000 claims description 6
- 208000004232 Enteritis Diseases 0.000 claims description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
- 241001478225 Riemerella Species 0.000 claims description 6
- 230000002757 inflammatory effect Effects 0.000 claims description 5
- 241000186359 Mycobacterium Species 0.000 claims description 4
- 206010030113 Oedema Diseases 0.000 claims description 4
- 241000194017 Streptococcus Species 0.000 claims description 4
- 241000282898 Sus scrofa Species 0.000 claims description 4
- 229940065181 bacillus anthracis Drugs 0.000 claims description 4
- 208000001848 dysentery Diseases 0.000 claims description 4
- 239000003921 oil Substances 0.000 claims description 4
- 241000283690 Bos taurus Species 0.000 claims description 3
- 241000283073 Equus caballus Species 0.000 claims description 3
- 201000000297 Erysipelas Diseases 0.000 claims description 3
- 241000186811 Erysipelothrix Species 0.000 claims description 3
- 206010034107 Pasteurella infections Diseases 0.000 claims description 3
- 241001494479 Pecora Species 0.000 claims description 3
- 208000006673 asthma Diseases 0.000 claims description 3
- 201000005115 pasteurellosis Diseases 0.000 claims description 3
- 239000000843 powder Substances 0.000 claims description 3
- 230000006269 (delayed) early viral mRNA transcription Effects 0.000 claims description 2
- 241000272814 Anser sp. Species 0.000 claims description 2
- 241000282836 Camelus dromedarius Species 0.000 claims description 2
- 241001112695 Clostridiales Species 0.000 claims description 2
- 241000272201 Columbiformes Species 0.000 claims description 2
- 241000238557 Decapoda Species 0.000 claims description 2
- 241000282326 Felis catus Species 0.000 claims description 2
- 241000286209 Phasianidae Species 0.000 claims description 2
- 206010039438 Salmonella Infections Diseases 0.000 claims description 2
- 241000191940 Staphylococcus Species 0.000 claims description 2
- 125000000217 alkyl group Chemical group 0.000 claims description 2
- 125000003118 aryl group Chemical group 0.000 claims description 2
- 239000002775 capsule Substances 0.000 claims description 2
- 239000008187 granular material Substances 0.000 claims description 2
- 229910052736 halogen Inorganic materials 0.000 claims description 2
- 125000005843 halogen group Chemical group 0.000 claims description 2
- 125000005616 oxoacid group Chemical group 0.000 claims description 2
- 230000002062 proliferating effect Effects 0.000 claims description 2
- 206010039447 salmonellosis Diseases 0.000 claims description 2
- 239000000725 suspension Substances 0.000 claims description 2
- 201000008827 tuberculosis Diseases 0.000 claims description 2
- 125000005313 fatty acid group Chemical group 0.000 claims 2
- 241000009328 Perro Species 0.000 claims 1
- 239000006072 paste Substances 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 36
- 230000004083 survival effect Effects 0.000 abstract description 30
- 230000000844 anti-bacterial effect Effects 0.000 abstract description 26
- 241000282414 Homo sapiens Species 0.000 abstract description 12
- 206010040047 Sepsis Diseases 0.000 abstract description 8
- 235000014113 dietary fatty acids Nutrition 0.000 abstract description 8
- 229930195729 fatty acid Natural products 0.000 abstract description 8
- 239000000194 fatty acid Substances 0.000 abstract description 8
- 208000013223 septicemia Diseases 0.000 abstract description 8
- 231100000331 toxic Toxicity 0.000 abstract description 7
- 230000002588 toxic effect Effects 0.000 abstract description 7
- 241000607522 Aeromonas sobria Species 0.000 abstract description 5
- 150000001413 amino acids Chemical class 0.000 abstract description 5
- 150000004665 fatty acids Chemical class 0.000 abstract description 4
- 244000052769 pathogen Species 0.000 abstract description 3
- 206010034674 peritonitis Diseases 0.000 abstract description 3
- 239000007859 condensation product Substances 0.000 abstract 1
- XJTMYVOVQZMMKX-KRWDZBQOSA-N ethyl (2s)-5-(diaminomethylideneamino)-2-(dodecanoylamino)pentanoate Chemical compound CCCCCCCCCCCC(=O)N[C@H](C(=O)OCC)CCCN=C(N)N XJTMYVOVQZMMKX-KRWDZBQOSA-N 0.000 description 183
- 241000699670 Mus sp. Species 0.000 description 47
- 229940079593 drug Drugs 0.000 description 41
- -1 hemotoxin Proteins 0.000 description 37
- 244000052616 bacterial pathogen Species 0.000 description 36
- 238000012360 testing method Methods 0.000 description 32
- 238000000034 method Methods 0.000 description 26
- 230000001580 bacterial effect Effects 0.000 description 25
- 230000012010 growth Effects 0.000 description 23
- TZCPCKNHXULUIY-RGULYWFUSA-N 1,2-distearoyl-sn-glycero-3-phosphoserine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(=O)OC[C@H](N)C(O)=O)OC(=O)CCCCCCCCCCCCCCCCC TZCPCKNHXULUIY-RGULYWFUSA-N 0.000 description 21
- ZWZWYGMENQVNFU-UHFFFAOYSA-N Glycerophosphorylserin Natural products OC(=O)C(N)COP(O)(=O)OCC(O)CO ZWZWYGMENQVNFU-UHFFFAOYSA-N 0.000 description 21
- 239000007864 aqueous solution Substances 0.000 description 21
- 235000013594 poultry meat Nutrition 0.000 description 20
- 229940088710 antibiotic agent Drugs 0.000 description 19
- 238000002474 experimental method Methods 0.000 description 18
- 239000003674 animal food additive Substances 0.000 description 16
- 210000000683 abdominal cavity Anatomy 0.000 description 15
- 241000699666 Mus <mouse, genus> Species 0.000 description 13
- 206010059866 Drug resistance Diseases 0.000 description 12
- 241000124008 Mammalia Species 0.000 description 12
- 230000003115 biocidal effect Effects 0.000 description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 11
- 230000002354 daily effect Effects 0.000 description 11
- 210000003743 erythrocyte Anatomy 0.000 description 11
- 239000000203 mixture Substances 0.000 description 11
- 239000000047 product Substances 0.000 description 11
- 239000000126 substance Substances 0.000 description 11
- 241000276707 Tilapia Species 0.000 description 10
- 238000009739 binding Methods 0.000 description 10
- 210000004027 cell Anatomy 0.000 description 10
- 229960005091 chloramphenicol Drugs 0.000 description 10
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 10
- 230000028161 membrane depolarization Effects 0.000 description 10
- 210000000056 organ Anatomy 0.000 description 10
- 239000013641 positive control Substances 0.000 description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- 235000019786 weight gain Nutrition 0.000 description 10
- JZNWSCPGTDBMEW-UHFFFAOYSA-N Glycerophosphorylethanolamin Natural products NCCOP(O)(=O)OCC(O)CO JZNWSCPGTDBMEW-UHFFFAOYSA-N 0.000 description 9
- 230000001976 improved effect Effects 0.000 description 9
- 230000002401 inhibitory effect Effects 0.000 description 9
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 description 9
- 230000004584 weight gain Effects 0.000 description 9
- 241000272517 Anseriformes Species 0.000 description 8
- 230000034994 death Effects 0.000 description 8
- 239000007788 liquid Substances 0.000 description 8
- 239000013642 negative control Substances 0.000 description 8
- 238000011160 research Methods 0.000 description 8
- 238000010171 animal model Methods 0.000 description 7
- 235000013330 chicken meat Nutrition 0.000 description 7
- 239000007850 fluorescent dye Substances 0.000 description 7
- 238000001727 in vivo Methods 0.000 description 7
- 235000016709 nutrition Nutrition 0.000 description 7
- 150000008104 phosphatidylethanolamines Chemical class 0.000 description 7
- 230000001225 therapeutic effect Effects 0.000 description 7
- 230000009105 vegetative growth Effects 0.000 description 7
- 206010018910 Haemolysis Diseases 0.000 description 6
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 6
- 235000001014 amino acid Nutrition 0.000 description 6
- 230000008859 change Effects 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- 239000002537 cosmetic Substances 0.000 description 6
- POULHZVOKOAJMA-UHFFFAOYSA-N dodecanoic acid Chemical compound CCCCCCCCCCCC(O)=O POULHZVOKOAJMA-UHFFFAOYSA-N 0.000 description 6
- 230000008588 hemolysis Effects 0.000 description 6
- 230000004060 metabolic process Effects 0.000 description 6
- 150000003904 phospholipids Chemical class 0.000 description 6
- 238000010186 staining Methods 0.000 description 6
- 229930182566 Gentamicin Natural products 0.000 description 5
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 5
- 108010093965 Polymyxin B Proteins 0.000 description 5
- 108010059993 Vancomycin Proteins 0.000 description 5
- 238000009395 breeding Methods 0.000 description 5
- 230000001488 breeding effect Effects 0.000 description 5
- 238000010790 dilution Methods 0.000 description 5
- 239000012895 dilution Substances 0.000 description 5
- 239000012530 fluid Substances 0.000 description 5
- 235000013305 food Nutrition 0.000 description 5
- 229960002518 gentamicin Drugs 0.000 description 5
- 210000002216 heart Anatomy 0.000 description 5
- 210000004185 liver Anatomy 0.000 description 5
- 210000004962 mammalian cell Anatomy 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 238000002156 mixing Methods 0.000 description 5
- 229920000024 polymyxin B Polymers 0.000 description 5
- 229960005266 polymyxin b Drugs 0.000 description 5
- 239000003755 preservative agent Substances 0.000 description 5
- 230000002335 preservative effect Effects 0.000 description 5
- 210000000952 spleen Anatomy 0.000 description 5
- 238000004448 titration Methods 0.000 description 5
- 230000001988 toxicity Effects 0.000 description 5
- 231100000419 toxicity Toxicity 0.000 description 5
- 229960003165 vancomycin Drugs 0.000 description 5
- MYPYJXKWCTUITO-LYRMYLQWSA-N vancomycin Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C2C=C3C=C1OC1=CC=C(C=C1Cl)[C@@H](O)[C@H](C(N[C@@H](CC(N)=O)C(=O)N[C@H]3C(=O)N[C@H]1C(=O)N[C@H](C(N[C@@H](C3=CC(O)=CC(O)=C3C=3C(O)=CC=C1C=3)C(O)=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)O2)=O)NC(=O)[C@@H](CC(C)C)NC)[C@H]1C[C@](C)(N)[C@H](O)[C@H](C)O1 MYPYJXKWCTUITO-LYRMYLQWSA-N 0.000 description 5
- MYPYJXKWCTUITO-UHFFFAOYSA-N vancomycin Natural products O1C(C(=C2)Cl)=CC=C2C(O)C(C(NC(C2=CC(O)=CC(O)=C2C=2C(O)=CC=C3C=2)C(O)=O)=O)NC(=O)C3NC(=O)C2NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(CC(C)C)NC)C(O)C(C=C3Cl)=CC=C3OC3=CC2=CC1=C3OC1OC(CO)C(O)C(O)C1OC1CC(C)(N)C(O)C(C)O1 MYPYJXKWCTUITO-UHFFFAOYSA-N 0.000 description 5
- 239000004475 Arginine Substances 0.000 description 4
- 241000167854 Bourreria succulenta Species 0.000 description 4
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 4
- 208000035240 Disease Resistance Diseases 0.000 description 4
- 241000192125 Firmicutes Species 0.000 description 4
- 241000283973 Oryctolagus cuniculus Species 0.000 description 4
- 238000009825 accumulation Methods 0.000 description 4
- 230000001154 acute effect Effects 0.000 description 4
- 239000004599 antimicrobial Substances 0.000 description 4
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 4
- 235000009697 arginine Nutrition 0.000 description 4
- 210000003567 ascitic fluid Anatomy 0.000 description 4
- 235000019693 cherries Nutrition 0.000 description 4
- 230000006806 disease prevention Effects 0.000 description 4
- 239000003640 drug residue Substances 0.000 description 4
- 230000005284 excitation Effects 0.000 description 4
- 239000005452 food preservative Substances 0.000 description 4
- 235000019249 food preservative Nutrition 0.000 description 4
- 210000001035 gastrointestinal tract Anatomy 0.000 description 4
- 238000007912 intraperitoneal administration Methods 0.000 description 4
- 231100000053 low toxicity Toxicity 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 230000035772 mutation Effects 0.000 description 4
- 239000002994 raw material Substances 0.000 description 4
- 230000002829 reductive effect Effects 0.000 description 4
- PESXGULMKCKJCC-UHFFFAOYSA-M sodium;4-methoxycarbonylphenolate Chemical compound [Na+].COC(=O)C1=CC=C([O-])C=C1 PESXGULMKCKJCC-UHFFFAOYSA-M 0.000 description 4
- 230000001954 sterilising effect Effects 0.000 description 4
- 238000004659 sterilization and disinfection Methods 0.000 description 4
- 231100000456 subacute toxicity Toxicity 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- GOZMBJCYMQQACI-UHFFFAOYSA-N 6,7-dimethyl-3-[[methyl-[2-[methyl-[[1-[3-(trifluoromethyl)phenyl]indol-3-yl]methyl]amino]ethyl]amino]methyl]chromen-4-one;dihydrochloride Chemical compound Cl.Cl.C=1OC2=CC(C)=C(C)C=C2C(=O)C=1CN(C)CCN(C)CC(C1=CC=CC=C11)=CN1C1=CC=CC(C(F)(F)F)=C1 GOZMBJCYMQQACI-UHFFFAOYSA-N 0.000 description 3
- FIZZUEJIOKEFFZ-UHFFFAOYSA-M C3-oxacyanine Chemical compound [I-].O1C2=CC=CC=C2[N+](CC)=C1C=CC=C1N(CC)C2=CC=CC=C2O1 FIZZUEJIOKEFFZ-UHFFFAOYSA-M 0.000 description 3
- VZBILKJHDPEENF-UHFFFAOYSA-M C3-thiacarbocyanine Chemical compound [I-].S1C2=CC=CC=C2[N+](CC)=C1C=CC=C1N(CC)C2=CC=CC=C2S1 VZBILKJHDPEENF-UHFFFAOYSA-M 0.000 description 3
- UGTJLJZQQFGTJD-UHFFFAOYSA-N Carbonylcyanide-3-chlorophenylhydrazone Chemical compound ClC1=CC=CC(NN=C(C#N)C#N)=C1 UGTJLJZQQFGTJD-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- AHLPHDHHMVZTML-BYPYZUCNSA-N L-Ornithine Chemical compound NCCC[C@H](N)C(O)=O AHLPHDHHMVZTML-BYPYZUCNSA-N 0.000 description 3
- AHLPHDHHMVZTML-UHFFFAOYSA-N Orn-delta-NH2 Natural products NCCCC(N)C(O)=O AHLPHDHHMVZTML-UHFFFAOYSA-N 0.000 description 3
- UTJLXEIPEHZYQJ-UHFFFAOYSA-N Ornithine Natural products OC(=O)C(C)CCCN UTJLXEIPEHZYQJ-UHFFFAOYSA-N 0.000 description 3
- 229960000723 ampicillin Drugs 0.000 description 3
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 3
- 230000002924 anti-infective effect Effects 0.000 description 3
- 229940124350 antibacterial drug Drugs 0.000 description 3
- 235000021053 average weight gain Nutrition 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
- 239000004202 carbamide Substances 0.000 description 3
- MLYYVTUWGNIJIB-BXKDBHETSA-N cefazolin Chemical compound S1C(C)=NN=C1SCC1=C(C(O)=O)N2C(=O)[C@@H](NC(=O)CN3N=NN=C3)[C@H]2SC1 MLYYVTUWGNIJIB-BXKDBHETSA-N 0.000 description 3
- CYDMQBQPVICBEU-UHFFFAOYSA-N chlorotetracycline Natural products C1=CC(Cl)=C2C(O)(C)C3CC4C(N(C)C)C(O)=C(C(N)=O)C(=O)C4(O)C(O)=C3C(=O)C2=C1O CYDMQBQPVICBEU-UHFFFAOYSA-N 0.000 description 3
- CYDMQBQPVICBEU-XRNKAMNCSA-N chlortetracycline Chemical compound C1=CC(Cl)=C2[C@](O)(C)[C@H]3C[C@H]4[C@H](N(C)C)C(O)=C(C(N)=O)C(=O)[C@@]4(O)C(O)=C3C(=O)C2=C1O CYDMQBQPVICBEU-XRNKAMNCSA-N 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 235000005911 diet Nutrition 0.000 description 3
- 230000037213 diet Effects 0.000 description 3
- 238000003113 dilution method Methods 0.000 description 3
- 239000000975 dye Substances 0.000 description 3
- 210000003527 eukaryotic cell Anatomy 0.000 description 3
- 238000000855 fermentation Methods 0.000 description 3
- 230000004151 fermentation Effects 0.000 description 3
- 238000000684 flow cytometry Methods 0.000 description 3
- 235000012055 fruits and vegetables Nutrition 0.000 description 3
- 238000003304 gavage Methods 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 210000003128 head Anatomy 0.000 description 3
- 230000036541 health Effects 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 210000003734 kidney Anatomy 0.000 description 3
- 230000002147 killing effect Effects 0.000 description 3
- 230000031700 light absorption Effects 0.000 description 3
- 210000004072 lung Anatomy 0.000 description 3
- 235000019198 oils Nutrition 0.000 description 3
- 229960003104 ornithine Drugs 0.000 description 3
- 239000002504 physiological saline solution Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 238000004321 preservation Methods 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 210000002345 respiratory system Anatomy 0.000 description 3
- 230000002000 scavenging effect Effects 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 230000000638 stimulation Effects 0.000 description 3
- 239000013589 supplement Substances 0.000 description 3
- 208000011580 syndromic disease Diseases 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 239000000273 veterinary drug Substances 0.000 description 3
- ODKSFYDXXFIFQN-BYPYZUCNSA-N L-arginine Chemical compound OC(=O)[C@@H](N)CCCN=C(N)N ODKSFYDXXFIFQN-BYPYZUCNSA-N 0.000 description 2
- 229930064664 L-arginine Natural products 0.000 description 2
- 235000014852 L-arginine Nutrition 0.000 description 2
- 239000005639 Lauric acid Substances 0.000 description 2
- 239000004100 Oxytetracycline Substances 0.000 description 2
- 244000046052 Phaseolus vulgaris Species 0.000 description 2
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 2
- 206010035148 Plague Diseases 0.000 description 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 2
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical group [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 241000282887 Suidae Species 0.000 description 2
- 239000004098 Tetracycline Substances 0.000 description 2
- 241000607479 Yersinia pestis Species 0.000 description 2
- 240000008042 Zea mays Species 0.000 description 2
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 2
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 239000004480 active ingredient Substances 0.000 description 2
- 231100000215 acute (single dose) toxicity testing Toxicity 0.000 description 2
- 231100000403 acute toxicity Toxicity 0.000 description 2
- 238000011047 acute toxicity test Methods 0.000 description 2
- 230000000845 anti-microbial effect Effects 0.000 description 2
- 206010003246 arthritis Diseases 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 230000003385 bacteriostatic effect Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 238000007664 blowing Methods 0.000 description 2
- 239000001569 carbon dioxide Substances 0.000 description 2
- 229910002092 carbon dioxide Inorganic materials 0.000 description 2
- 229960001139 cefazolin Drugs 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 229960004475 chlortetracycline Drugs 0.000 description 2
- 235000019365 chlortetracycline Nutrition 0.000 description 2
- 230000001684 chronic effect Effects 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 230000005757 colony formation Effects 0.000 description 2
- 235000005822 corn Nutrition 0.000 description 2
- 231100000135 cytotoxicity Toxicity 0.000 description 2
- 230000003013 cytotoxicity Effects 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 239000003651 drinking water Substances 0.000 description 2
- 235000020188 drinking water Nutrition 0.000 description 2
- 238000004043 dyeing Methods 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 230000003203 everyday effect Effects 0.000 description 2
- 210000002950 fibroblast Anatomy 0.000 description 2
- 238000011010 flushing procedure Methods 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 230000002496 gastric effect Effects 0.000 description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
- 239000010931 gold Substances 0.000 description 2
- 229910052737 gold Inorganic materials 0.000 description 2
- 230000002949 hemolytic effect Effects 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 239000007928 intraperitoneal injection Substances 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 230000006996 mental state Effects 0.000 description 2
- 230000002503 metabolic effect Effects 0.000 description 2
- 239000002207 metabolite Substances 0.000 description 2
- 229960003476 methylparaben sodium Drugs 0.000 description 2
- 239000002324 mouth wash Substances 0.000 description 2
- 229940051866 mouthwash Drugs 0.000 description 2
- 231100000252 nontoxic Toxicity 0.000 description 2
- 230000003000 nontoxic effect Effects 0.000 description 2
- 229960000625 oxytetracycline Drugs 0.000 description 2
- IWVCMVBTMGNXQD-PXOLEDIWSA-N oxytetracycline Chemical compound C1=CC=C2[C@](O)(C)[C@H]3[C@H](O)[C@H]4[C@H](N(C)C)C(O)=C(C(N)=O)C(=O)[C@@]4(O)C(O)=C3C(=O)C2=C1O IWVCMVBTMGNXQD-PXOLEDIWSA-N 0.000 description 2
- 235000019366 oxytetracycline Nutrition 0.000 description 2
- 210000001236 prokaryotic cell Anatomy 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 210000005084 renal tissue Anatomy 0.000 description 2
- 239000002453 shampoo Substances 0.000 description 2
- 235000010268 sodium methyl p-hydroxybenzoate Nutrition 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- 239000004455 soybean meal Substances 0.000 description 2
- 230000000475 sunscreen effect Effects 0.000 description 2
- 239000000516 sunscreening agent Substances 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 230000002195 synergetic effect Effects 0.000 description 2
- IWVCMVBTMGNXQD-UHFFFAOYSA-N terramycin dehydrate Natural products C1=CC=C2C(O)(C)C3C(O)C4C(N(C)C)C(O)=C(C(N)=O)C(=O)C4(O)C(O)=C3C(=O)C2=C1O IWVCMVBTMGNXQD-UHFFFAOYSA-N 0.000 description 2
- 229960002180 tetracycline Drugs 0.000 description 2
- 235000019364 tetracycline Nutrition 0.000 description 2
- 229930101283 tetracycline Natural products 0.000 description 2
- 150000003522 tetracyclines Chemical class 0.000 description 2
- 229940124597 therapeutic agent Drugs 0.000 description 2
- 231100000155 toxicity by organ Toxicity 0.000 description 2
- 230000007675 toxicity by organ Effects 0.000 description 2
- 231100000041 toxicology testing Toxicity 0.000 description 2
- 239000001974 tryptic soy broth Substances 0.000 description 2
- 108010050327 trypticase-soy broth Proteins 0.000 description 2
- 210000002700 urine Anatomy 0.000 description 2
- 230000001018 virulence Effects 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- SGKRLCUYIXIAHR-AKNGSSGZSA-N (4s,4ar,5s,5ar,6r,12ar)-4-(dimethylamino)-1,5,10,11,12a-pentahydroxy-6-methyl-3,12-dioxo-4a,5,5a,6-tetrahydro-4h-tetracene-2-carboxamide Chemical compound C1=CC=C2[C@H](C)[C@@H]([C@H](O)[C@@H]3[C@](C(O)=C(C(N)=O)C(=O)[C@H]3N(C)C)(O)C3=O)C3=C(O)C2=C1O SGKRLCUYIXIAHR-AKNGSSGZSA-N 0.000 description 1
- 229940100555 2-methyl-4-isothiazolin-3-one Drugs 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 206010003591 Ataxia Diseases 0.000 description 1
- SPFYMRJSYKOXGV-UHFFFAOYSA-N Baytril Chemical compound C1CN(CC)CCN1C(C(=C1)F)=CC2=C1C(=O)C(C(O)=O)=CN2C1CC1 SPFYMRJSYKOXGV-UHFFFAOYSA-N 0.000 description 1
- 239000005711 Benzoic acid Substances 0.000 description 1
- 241000272834 Cairina moschata Species 0.000 description 1
- 240000001548 Camellia japonica Species 0.000 description 1
- 108010065152 Coagulase Proteins 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 206010011224 Cough Diseases 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- 208000002064 Dental Plaque Diseases 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 241000588914 Enterobacter Species 0.000 description 1
- 101710146739 Enterotoxin Proteins 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241001360526 Escherichia coli ATCC 25922 Species 0.000 description 1
- 241000628997 Flos Species 0.000 description 1
- 206010016952 Food poisoning Diseases 0.000 description 1
- 208000019331 Foodborne disease Diseases 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 102000001554 Hemoglobins Human genes 0.000 description 1
- 108010054147 Hemoglobins Proteins 0.000 description 1
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 229920001202 Inulin Polymers 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 1
- 108010014603 Leukocidins Proteins 0.000 description 1
- 244000245109 Lindera glauca Species 0.000 description 1
- 235000001577 Lindera glauca Nutrition 0.000 description 1
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 1
- 201000009906 Meningitis Diseases 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 206010058667 Oral toxicity Diseases 0.000 description 1
- 241000276701 Oreochromis mossambicus Species 0.000 description 1
- 235000019482 Palm oil Nutrition 0.000 description 1
- 206010071301 Perihepatitis Diseases 0.000 description 1
- 208000007893 Salpingitis Diseases 0.000 description 1
- 206010070834 Sensitisation Diseases 0.000 description 1
- 206010058556 Serositis Diseases 0.000 description 1
- 206010072170 Skin wound Diseases 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 206010044565 Tremor Diseases 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000004182 Tylosin Substances 0.000 description 1
- 229930194936 Tylosin Natural products 0.000 description 1
- 239000004904 UV filter Substances 0.000 description 1
- 206010048038 Wound infection Diseases 0.000 description 1
- 230000003187 abdominal effect Effects 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 229960003022 amoxicillin Drugs 0.000 description 1
- LSQZJLSUYDQPKJ-NJBDSQKTSA-N amoxicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=C(O)C=C1 LSQZJLSUYDQPKJ-NJBDSQKTSA-N 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000000842 anti-protozoal effect Effects 0.000 description 1
- 230000002421 anti-septic effect Effects 0.000 description 1
- 230000010100 anticoagulation Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 239000003904 antiprotozoal agent Substances 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- 210000001367 artery Anatomy 0.000 description 1
- 230000001174 ascending effect Effects 0.000 description 1
- 239000000022 bacteriostatic agent Substances 0.000 description 1
- 235000010233 benzoic acid Nutrition 0.000 description 1
- 238000005842 biochemical reaction Methods 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 238000010241 blood sampling Methods 0.000 description 1
- 238000002815 broth microdilution Methods 0.000 description 1
- 125000001589 carboacyl group Chemical group 0.000 description 1
- 239000003093 cationic surfactant Substances 0.000 description 1
- 230000003833 cell viability Effects 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
- 235000013351 cheese Nutrition 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- MRUAUOIMASANKQ-UHFFFAOYSA-N cocamidopropyl betaine Chemical compound CCCCCCCCCCCC(=O)NCCC[N+](C)(C)CC([O-])=O MRUAUOIMASANKQ-UHFFFAOYSA-N 0.000 description 1
- 229940073507 cocamidopropyl betaine Drugs 0.000 description 1
- 239000003240 coconut oil Substances 0.000 description 1
- 235000019864 coconut oil Nutrition 0.000 description 1
- 235000018597 common camellia Nutrition 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 230000003750 conditioning effect Effects 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 229920006037 cross link polymer Polymers 0.000 description 1
- 238000002784 cytotoxicity assay Methods 0.000 description 1
- 231100000263 cytotoxicity test Toxicity 0.000 description 1
- 230000002999 depolarising effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- PXEDJBXQKAGXNJ-QTNFYWBSSA-L disodium L-glutamate Chemical compound [Na+].[Na+].[O-]C(=O)[C@@H](N)CCC([O-])=O PXEDJBXQKAGXNJ-QTNFYWBSSA-L 0.000 description 1
- 229940079779 disodium cocoyl glutamate Drugs 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- ILRSCQWREDREME-UHFFFAOYSA-N dodecanamide Chemical compound CCCCCCCCCCCC(N)=O ILRSCQWREDREME-UHFFFAOYSA-N 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 229960003722 doxycycline Drugs 0.000 description 1
- 238000001647 drug administration Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 239000000428 dust Substances 0.000 description 1
- 239000008157 edible vegetable oil Substances 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 206010014801 endophthalmitis Diseases 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 229960000740 enrofloxacin Drugs 0.000 description 1
- 231100000655 enterotoxin Toxicity 0.000 description 1
- 239000000147 enterotoxin Substances 0.000 description 1
- BEFDCLMNVWHSGT-UHFFFAOYSA-N ethenylcyclopentane Chemical compound C=CC1CCCC1 BEFDCLMNVWHSGT-UHFFFAOYSA-N 0.000 description 1
- 125000004494 ethyl ester group Chemical group 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 238000009313 farming Methods 0.000 description 1
- 230000037406 food intake Effects 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000037308 hair color Effects 0.000 description 1
- 231100000767 hemotoxin Toxicity 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 206010019692 hepatic necrosis Diseases 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 150000004677 hydrates Chemical class 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000002458 infectious effect Effects 0.000 description 1
- 239000000543 intermediate Substances 0.000 description 1
- 239000013067 intermediate product Substances 0.000 description 1
- JYJIGFIDKWBXDU-MNNPPOADSA-N inulin Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)OC[C@]1(OC[C@]2(OC[C@]3(OC[C@]4(OC[C@]5(OC[C@]6(OC[C@]7(OC[C@]8(OC[C@]9(OC[C@]%10(OC[C@]%11(OC[C@]%12(OC[C@]%13(OC[C@]%14(OC[C@]%15(OC[C@]%16(OC[C@]%17(OC[C@]%18(OC[C@]%19(OC[C@]%20(OC[C@]%21(OC[C@]%22(OC[C@]%23(OC[C@]%24(OC[C@]%25(OC[C@]%26(OC[C@]%27(OC[C@]%28(OC[C@]%29(OC[C@]%30(OC[C@]%31(OC[C@]%32(OC[C@]%33(OC[C@]%34(OC[C@]%35(OC[C@]%36(O[C@@H]%37[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O%37)O)[C@H]([C@H](O)[C@@H](CO)O%36)O)[C@H]([C@H](O)[C@@H](CO)O%35)O)[C@H]([C@H](O)[C@@H](CO)O%34)O)[C@H]([C@H](O)[C@@H](CO)O%33)O)[C@H]([C@H](O)[C@@H](CO)O%32)O)[C@H]([C@H](O)[C@@H](CO)O%31)O)[C@H]([C@H](O)[C@@H](CO)O%30)O)[C@H]([C@H](O)[C@@H](CO)O%29)O)[C@H]([C@H](O)[C@@H](CO)O%28)O)[C@H]([C@H](O)[C@@H](CO)O%27)O)[C@H]([C@H](O)[C@@H](CO)O%26)O)[C@H]([C@H](O)[C@@H](CO)O%25)O)[C@H]([C@H](O)[C@@H](CO)O%24)O)[C@H]([C@H](O)[C@@H](CO)O%23)O)[C@H]([C@H](O)[C@@H](CO)O%22)O)[C@H]([C@H](O)[C@@H](CO)O%21)O)[C@H]([C@H](O)[C@@H](CO)O%20)O)[C@H]([C@H](O)[C@@H](CO)O%19)O)[C@H]([C@H](O)[C@@H](CO)O%18)O)[C@H]([C@H](O)[C@@H](CO)O%17)O)[C@H]([C@H](O)[C@@H](CO)O%16)O)[C@H]([C@H](O)[C@@H](CO)O%15)O)[C@H]([C@H](O)[C@@H](CO)O%14)O)[C@H]([C@H](O)[C@@H](CO)O%13)O)[C@H]([C@H](O)[C@@H](CO)O%12)O)[C@H]([C@H](O)[C@@H](CO)O%11)O)[C@H]([C@H](O)[C@@H](CO)O%10)O)[C@H]([C@H](O)[C@@H](CO)O9)O)[C@H]([C@H](O)[C@@H](CO)O8)O)[C@H]([C@H](O)[C@@H](CO)O7)O)[C@H]([C@H](O)[C@@H](CO)O6)O)[C@H]([C@H](O)[C@@H](CO)O5)O)[C@H]([C@H](O)[C@@H](CO)O4)O)[C@H]([C@H](O)[C@@H](CO)O3)O)[C@H]([C@H](O)[C@@H](CO)O2)O)[C@@H](O)[C@H](O)[C@@H](CO)O1 JYJIGFIDKWBXDU-MNNPPOADSA-N 0.000 description 1
- 229940029339 inulin Drugs 0.000 description 1
- 238000003973 irrigation Methods 0.000 description 1
- 239000002085 irritant Substances 0.000 description 1
- 231100000021 irritant Toxicity 0.000 description 1
- 230000007794 irritation Effects 0.000 description 1
- 238000000111 isothermal titration calorimetry Methods 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 229940048848 lauryl glucoside Drugs 0.000 description 1
- 231100001231 less toxic Toxicity 0.000 description 1
- 231100000149 liver necrosis Toxicity 0.000 description 1
- 231100001252 long-term toxicity Toxicity 0.000 description 1
- 239000006210 lotion Substances 0.000 description 1
- 208000004396 mastitis Diseases 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 235000013372 meat Nutrition 0.000 description 1
- 235000013622 meat product Nutrition 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- VUZPPFZMUPKLLV-UHFFFAOYSA-N methane;hydrate Chemical compound C.O VUZPPFZMUPKLLV-UHFFFAOYSA-N 0.000 description 1
- BEGLCMHJXHIJLR-UHFFFAOYSA-N methylisothiazolinone Chemical group CN1SC=CC1=O BEGLCMHJXHIJLR-UHFFFAOYSA-N 0.000 description 1
- 230000003641 microbiacidal effect Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 229940124561 microbicide Drugs 0.000 description 1
- 239000002855 microbicide agent Substances 0.000 description 1
- 230000008722 morphological abnormality Effects 0.000 description 1
- 210000000214 mouth Anatomy 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 210000000651 myofibroblast Anatomy 0.000 description 1
- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 description 1
- 239000002674 ointment Substances 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 231100000418 oral toxicity Toxicity 0.000 description 1
- 239000005416 organic matter Substances 0.000 description 1
- LSQZJLSUYDQPKJ-UHFFFAOYSA-N p-Hydroxyampicillin Natural products O=C1N2C(C(O)=O)C(C)(C)SC2C1NC(=O)C(N)C1=CC=C(O)C=C1 LSQZJLSUYDQPKJ-UHFFFAOYSA-N 0.000 description 1
- 239000002540 palm oil Substances 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 230000007918 pathogenicity Effects 0.000 description 1
- 238000010827 pathological analysis Methods 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- 208000008494 pericarditis Diseases 0.000 description 1
- 239000008194 pharmaceutical composition Substances 0.000 description 1
- 229930010796 primary metabolite Natural products 0.000 description 1
- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 150000004671 saturated fatty acids Chemical class 0.000 description 1
- 238000001878 scanning electron micrograph Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 230000008313 sensitization Effects 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229940083542 sodium Drugs 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 235000010199 sorbic acid Nutrition 0.000 description 1
- 239000004334 sorbic acid Substances 0.000 description 1
- 229940075582 sorbic acid Drugs 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 125000005472 straight-chain saturated fatty acid group Chemical group 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- BDHFUVZGWQCTTF-UHFFFAOYSA-M sulfonate Chemical compound [O-]S(=O)=O BDHFUVZGWQCTTF-UHFFFAOYSA-M 0.000 description 1
- 238000000352 supercritical drying Methods 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- 229940034610 toothpaste Drugs 0.000 description 1
- 239000000606 toothpaste Substances 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000017105 transposition Effects 0.000 description 1
- 150000003628 tricarboxylic acids Chemical class 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 229960004059 tylosin Drugs 0.000 description 1
- 235000019375 tylosin Nutrition 0.000 description 1
- WBPYTXDJUQJLPQ-VMXQISHHSA-N tylosin Chemical compound O([C@@H]1[C@@H](C)O[C@H]([C@@H]([C@H]1N(C)C)O)O[C@@H]1[C@@H](C)[C@H](O)CC(=O)O[C@@H]([C@H](/C=C(\C)/C=C/C(=O)[C@H](C)C[C@@H]1CC=O)CO[C@H]1[C@@H]([C@H](OC)[C@H](O)[C@@H](C)O1)OC)CC)[C@H]1C[C@@](C)(O)[C@@H](O)[C@H](C)O1 WBPYTXDJUQJLPQ-VMXQISHHSA-N 0.000 description 1
- 230000004143 urea cycle Effects 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 210000001835 viscera Anatomy 0.000 description 1
- 230000003442 weekly effect Effects 0.000 description 1
- 206010048282 zoonosis Diseases 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
- A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
- A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
- A61K31/223—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin of alpha-aminoacids
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/142—Amino acids; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/195—Antibiotics
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Polymers & Plastics (AREA)
- Animal Husbandry (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Zoology (AREA)
- Pharmacology & Pharmacy (AREA)
- Emergency Medicine (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
Abstract
The present invention relates to a kind of novel veterinary antibacterial agents, the antibacterial agent is comprising the antibacterial agent from fatty acid and the condensation product for being esterified binary amino acid, it can prevent and treat the duckling Riemerella anatipestifer disease as caused by riemerella anatipestifer, the mouse peritoneum inflammation disease as caused by Escherichia coli and staphylococcus aureus can be treated, the fish septicemia caused by Aeromonas sobria can be prevented and treated.The antibacterial agent can effectively inhibit or kill bacterium, the risk of poultry, domestic animal and aquatic livestock bacterial infection can be greatly lowered, and promote the survival rate of infection pathogen animal.Lauroyl arginine ethyl ester in human body and animal body can safe disposal, toxic side effect is small, avoid the generation of the environment drug-fast bacteria caused by being discharged into environment due to remaining antibacterial agent, while reaching effective antibacterial effect to environment negatively affect it is small.
Description
The invention requires Chinese patent application 201711071006.8, named as lauroyl arginine
Ethyl esters and derivatives thereof as veterinary agentsThe application of priority to the use of microbial agents.
Technical Field
The invention relates to application of a compound lauroyl arginine ethyl ester in preparing veterinary antibacterial agents, feed additives or feed nutritional energy substances.
Background
Riemerella anatipestifer disease is a contact infectious disease caused by Riemerella Anatipestifer (RA), and is also called duck infectious serositis, duck septicemia, duck plague syndrome, pasteurellosis anatipestifer disease and the like. The ducklings are mostly seen in 1-8 weeks old, and the ducklings 2-4 weeks old are most susceptible. Acute or chronic septicemia is mainly manifested by eye and nose secretion increase, asthma, cough, diarrhea, ataxia and head and neck tremor, and head and neck distortion in a few chronic cases. The pathological changes are characterized by cellulose pericarditis, perihepatitis, air sacculitis, meningitis and arthritis appearing in partial cases, and the diseases often cause a large amount of morbidity and mortality of ducklings. The morbidity of the disease can reach more than 90 percent, and the mortality is related to the day age of the diseased duck, the virulence of the strain and adverse stress factors, and can reach 75 percent at most. Besides death of 1-8 weeks old ducks, salpingitis can be caused, laying rate of adult ducks is reduced, growth of adult ducks is retarded, and great economic loss is caused to farmers. Since the first report in 1982, the disease has become one of the most common bacterial diseases endangering the meat duck breeding industry. Under natural conditions, the disease occurs all the year round. Mainly through contaminated feed, drinking water, dust, spray and the like, enter the body of the poultry through respiratory tract, digestive tract or skin wounds (especially webbed skin) to cause diseases. Various kinds of ducks such as Beijing duck, cherry valley duck, Digao duck, Muscovy duck, sheldrake and the like can be infected and diseased. 48 duck farms in Jiangsu province are randomly spot-checked from 7 to 8 months in 2012, wherein 45 duck farms have suspected riemerella anatipestifer disease cases, and even 12 duck farms have twice outbreaks of the disease during the spot-check. In addition, if a certain duck farm suffers from the disease, the disease can be developed in the surrounding duck farms successively, so that the disease can be seen to cause serious damage to the duck farming industry.
Staphylococcus aureus is a generic term for the different diseases or types of diseases in many animals caused by Staphylococcus aureus. The pathogenic staphylococcus aureus can cause the diseases of various poultry and livestock, young livestock and poultry are most susceptible to the diseases and are mostly infected through digestive tracts, chickens can also be infected through respiratory tracts, and common symptoms comprise diarrhea, enteritis, liver necrosis and the like. Suppurative diseases are usually caused, and mastitis, arthritis, wound infection, septicemia and the like of animals are mainly caused; the other is toxic diseases, and the feed polluted by pathogenic bacteria can cause toxic enteritis of animals and toxin shock syndrome of human beings, etc. The pathogenicity of the staphylococcus aureus is mainly determined by virulence pathogenic factors generated by the staphylococcus aureus, and mainly comprises plasma coagulase, enterotoxin, heat-resistant nuclease, hemotoxin, leukocidin and the like.
Colibacillosis refers to the generic term for the different diseases or types of diseases in many animals caused by pathogenic E.coli. The pathogenic colibacilli and non-pathogenic colibacillus normally inhabiting human and animal intestinal tracts are not different in morphology, staining reaction, culture characteristic, biochemical reaction and the like, but have different antigen structures. Pathogenic Escherichia coli can cause diseases of various poultry and livestock, such as pig, cattle, sheep, horse, chicken, rabbit, etc., and young livestock and poultry are most susceptible to the diseases and are mostly infected through digestive tract, and chickens can also be infected through respiratory tract. The colibacillosis of pigs is also different in clinical manifestations of piglets according to the growth period of piglets and the serotype of pathogenic bacteria, and can be divided into yellow dysentery type, white dysentery type and edema type. When yellow scours occur to piglets, the disease death rate is high and can reach 100% in some piglets, which is more than 90% of the normal piglets and one litter of piglets; the incidence rate of white diarrhea is 30-80%; the incidence rate of edema disease is 10-35%. Colibacillosis in chicks is commonly manifested by acute septicemia, vitelline peritonitis, ophthalmia, ballonflam head syndrome, and the like. The morbidity can reach 30-60 percent, and the fatality rate can reach 100 percent.
Aeromonas sobria is a gram-negative facultative anaerobic bacterium, is usually present in various water environments and soil environments, and is a zoonosis pathogenic bacterium. Aeromonas sobria can produce various pathogenic factors such as lysin, ectoenzyme and the like, and can cause aquatic animals to suffer from septicemia, so that the aquatic animals die, and the economic basis of aquaculture is seriously influenced. In addition, pathogenic bacteria can infect people through aquatic products, and the patient can have symptoms such as diarrhea and the like and even develop food poisoning or septicemia.
Besides improving the feeding conditions, the application of antibiotics is a main measure for preventing and treating bacterial diseases of livestock, poultry and aquatic products. However, in recent years, common antibiotics such as aureomycin, oxytetracycline, tetracycline, chloramphenicol and the like are widely used in livestock and poultry breeding industry due to the disease-resistant and growth-promoting effects of the antibiotics. According to statistics, the annual production of antibiotic raw materials in China is about 21 ten thousand tons, and 9.7 ten thousand tons of antibiotic are used in livestock and poultry breeding industry and account for 46.1 percent of the total production. Improper antibiotic use, no guarantee of medicine quality, incomplete supervision and non-strict medication regulations lead to the aggravation of antibiotic abuse, so that a plurality of unreasonable and serious problems are generated, such as the generation of bacterial drug resistance, the reduction of animal organism immunity, the medicine residue of meat products and the like, and the health of human beings is directly harmed. According to researches, about 75% of antibiotics cannot be absorbed and metabolized by human or animal bodies, part of the antibiotics can remain in the bodies, and 20% -50% of live chicken or frozen chicken tissues can detect the antibiotic residues; the residual antibiotics can also enter the environment along with excrement, some antibiotics can directly enter a river channel and influence the drinking water safety of downstream residents, and the residual antibiotics of the livestock and poultry industry such as oxytetracycline, tetracycline, doxycycline, amoxicillin, aureomycin and the like are detected in tap water of many residents in rivers and cities in China. In recent years, the urine of 1000 children in Shanghai is researched to detect antibiotics, and a plurality of veterinary antibiotics (such as tylosin, chlorotetracycline, enrofloxacin and the like) which are only used in the breeding industry are detected in 58% of urine samples. More importantly, the residual antibiotics can naturally select the microorganisms in the environment or induce the genetic mutation of the microorganisms, and the bacteria with drug resistance can survive and continue to breed more drug-resistant bacteria. In addition, the bacteria can also transmit own drug-resistant genes to other microorganisms of different species and genera by means of conjugation, transformation, transduction, transposition and the like so as to obtain drug resistance. This makes the drug-resistant bacteria enriched in the environment, and the contaminated soil and water source are very easy to cause diseases and accelerate the diffusion of drug-resistant bacteria once contacted by human or livestock. Veterinary drugs and feed additives mainly include preservatives, dust-proofing agents, antioxidants, antiprotozoal drugs, and the like, in addition to illegal addition of antibiotics or antibacterial agents. The substances can bring harm to the health of livestock and poultry and human bodies due to long-term use or improper use.
Therefore, in the cultivation of aquatic products, livestock and poultry, especially in the cultivation of poultry (such as chicken and duck), there is an urgent need for an antibacterial agent which can be rapidly sterilized, is easily degraded in vivo, has no residue, can effectively prevent the occurrence and spread of diseases in the cultivation industry, and can avoid the influence of similar antibiotic residues on human body and environment.
Lauroyl arginine Ethyl ester (LAE) is an organic matter formed by condensing fatty acid and dibasic amino acid, is a white hygroscopic solid, is stable in chemical property within the range of pH 3-7, has a melting point of 50-58 ℃, can be dispersed in 1kg of water at the temperature of 247g, has a distribution coefficient of more than 10 in water and oil, and is mainly in a water phase. Researches find that the lauroyl arginine ethyl ester LAE has the characteristics of strong antibacterial capability, low biological toxicity, good in vivo metabolism effect and high environmental compatibility. The most representative characteristic is that no residue is left in the metabolism of lauroyl arginine ethyl ester, and related researches show that the lauroyl arginine ethyl ester can be rapidly and naturally metabolized in human bodies and animal bodies to generate lauric acid and arginine, and further be metabolized into ornithine, urea, carbon dioxide and water. All primary metabolites and final metabolites produced during the metabolism of lauroyl arginine ethyl ester are non-toxic and harmless, and are the same as the metabolites of food ingested daily by humans and animals in the body.
For example, Chinese patent application CN201710056593, entitled "a fruit and vegetable preservative and a preparation method and application thereof" discloses a composition taking lauroyl arginine ethyl ester hydrochloride and sodium methyl paraben as main active ingredients to be used as the fruit and vegetable preservative, which can effectively inhibit the growth of bacteria causing fruit and vegetable rot. However, the single bacteriostatic effect of the high-concentration methyl paraben sodium (2000 mug/ml) is stronger than that of the low-concentration LAE (1000 mug/ml) because the high-concentration methyl paraben sodium has a phenolic hydroxyl structure and the antibacterial performance is far stronger than that of benzoic acid and sorbic acid, so that on the premise of ensuring the preservative performance, the method definitely indicates that the use of the sodium methyl paraben instead of the LAE is beneficial to reducing the dosage cost of the preservative.
Chinese patent application CN201510748675, entitled "method for inhibiting alcohol fermentation contaminating microorganisms by using lauroyl arginine ethyl ester" discloses a method for inhibiting alcohol fermentation contaminating microorganisms by using lauroyl arginine ethyl ester, which comprises adding LAE and salt compounds thereof into fermentation liquor of saccharomyces cerevisiae at a concentration of less than 50 μ g/ml, and can effectively inhibit the growth of lactic acid bacteria and control the growth of other contaminating microorganisms. However, this bacteriostatic slightly affects yeast growth to some extent and results in a 0.6% reduction in alcohol production.
Chinese patent application CN201610466729, entitled "a mild infant shampoo and bath bubble" discloses a mild infant shampoo and bath bubble, which is prepared by selecting disodium cocoyl glutamate, cocamidopropyl betaine, sodium hydroxypropyl lauryl glucoside crosslinked polymer sulfonate as surfactant system, selecting camellia seed oil, α dextran oligosaccharide/inulin complex as conditioning component, and flos Chrysanthemi Indici extract and lauroyl arginine ethyl ester HCl as antiseptic system, wherein the raw materials cooperate with each other, and the bubble has good cleaning effect, and is mild and non-irritant.
chinese patent application CN201280073013, entitled "synergistic antimicrobial agent", discloses the production of more effective antimicrobial agents and food preservatives by combining an effective amount of an N- α -long chain alkanoyl dibasic amino acid alkyl ester salt with a glycerol mono fatty acid ester to provide a synergistic antimicrobial composition, while chinese patent application CN200810131638, entitled "microbicide composition", discloses the use of a composition of methylisothiazolinone and LAE for the preparation of antimicrobial agents and food preservatives.
Chinese patent application CN201280027864, entitled "cosmetic or dermatological sunscreen formulation with improved water resistance", discloses the use of LAE for the preparation of a cosmetic or dermatological sunscreen formulation comprising, in addition to a UV filter, the emulsifier polyglycerol-10 stearate.
As the closest prior art, chinese patent application CN200580051259, entitled "preservation system comprising cationic surfactant", discloses for the first time the use of LAE and its hydrochloride in preservation systems, which system comprising 0.2g/kg LAE is added in food, cosmetics to play a role of preservation. The invention researches the antibacterial mechanism of the LAE and provides the application of the LAE in the preservative action of foods, cosmetics and the like, so that the US food safety agency approves lauroyl arginine ethyl ester for food preservatives in 2005; the european union food safety agency, australia and new zealand in 2012 also approved lauroyl arginine ethyl ester for use as a food preservative. Meanwhile, in view of the application of the invention in cosmetics for the first time, the subsequent research finds that the lauroyl arginine ethyl ester can be used in products in oral care (such as US20100330136A1, EP2361606A2 and EP231603A2) such as mouthwash, toothpaste and the like, can effectively inhibit the formation of dental plaque in the oral cavity, is compatible with other chemical components in the mouthwash and has stable chemical properties; lauroyl arginine ethyl ester can be used in cosmetic products with topical therapeutic effect, which have the following characteristics: antibacterial effect, low toxicity, no sensitization, and no irritation to skin. Currently, researchers are developing hand lotions for cleansing and bacteriostatic agents for application to the skin surface.
On the other hand, research shows that the LAE is rapidly degraded in vivo, the lauroyl amide bond or the ester bond in the LAE is broken, the lauric acid part or the ethanol part is removed to form arginine ethanol ester or lauroyl amide arginine, and the ethanol part or the lauric acid part is removed respectively to form the same intermediate product L-arginine. Lauric acid produced by the metabolic process is also the main constituent of fatty acid of edible oil such as palm oil, lindera glauca oil, coconut oil and the like. Arginine is one of 20 amino acids constituting protein, exists in foods such as nut, cheese, fish and the like, belongs to the large category of amino acids, amino acid salts and analogues thereof according to a 2045 document, namely a feed additive variety catalog (2013) published by the ministry of agriculture of the people's republic of China, and is a feed additive for providing energy. In addition, L-arginine can be further hydrolyzed into ornithine and urea, wherein the ornithine can be synthesized into organic matters through a urea cycle and a citric acid (tricarboxylic acid) cycle, and finally decomposed into urea and carbon dioxide to be discharged out of the body. That is, the metabolic intermediates or end products produced by LAE during metabolism in animals are not only safe and non-toxic, but also generate a large amount of energy substances. Therefore, the LAE can play the role of antibiosis and providing metabolic energy when being applied to feed addition, thereby being beneficial to the health and the nutritional growth of animals.
Disclosure of Invention
In summary, the prior inventions do not teach how to use single lauroyl arginine ethyl ester (LAE) and its derivatives or hydrate components as antibacterial drugs or feed additives for livestock and aquatic products, nor disclose a suitable concentration of lauroyl arginine ethyl ester (LAE) and its derivatives or hydrates as antibacterial drugs or feed additives. Therefore, the lauroyl arginine ethyl ester and the derivative or hydrate (preferably LAE) thereof are firstly used in the research of antibacterial drugs for livestock, poultry and aquatic products, and the appropriate use concentration is determined through experiments, so that the antibacterial and disease-preventing effects are achieved, meanwhile, the negative effect on the environment is small, the toxic and side effects are low, and the serious bacterial drug resistance effect caused by the abuse of antibiotics in the livestock breeding industry is relieved. On the other hand, on the basis of the above research, the LAE component is used as a feed additive for the production of livestock, poultry and aquatic products. And finally, under the condition of ensuring the antibacterial effect of the LAE, respectively analyzing the influence of the LAE serving as an energy substance in different antibacterial feeds on the nutritional growth of aquatic animals of the livestock, so as to determine the proper proportion for simultaneously realizing the antibacterial and disease prevention and providing energy nutrition.
The invention aims to provide a compound lauroyl arginine ethyl ester (LAE) shown as a formula (I) and a derivative thereof, or a hydrate or a pharmaceutically acceptable salt thereof, and an application of the compound lauroyl arginine ethyl ester (LAE) in preparing a medicament for treating or preventing diseases of livestock and aquatic animals caused by pathogenic microorganisms.
The invention also provides a pharmaceutical composition, which comprises the lauroyl arginine ethyl ester compound (LAE compound) shown in the formula (I) or a hydrate or pharmaceutically acceptable salt thereof.
Wherein,
x is halogen or HSO4(ii) a Preferably, Br, Cl or HSO4；
R1Is a straight chain saturated fatty acid radical containing 8 to 14 carbon atoms, or an oxyacid radical containing 8 to 14 carbon atoms; preferably, a straight chain oxo acid group containing 12 carbon atoms;
R2is a linear fatty acid radical having from 1 to 18 carbon atoms, or a branched fatty acid radical having from 1 to 18 carbon atoms, orAn aromatic group of 1 to 18 carbon atoms, or a straight chain alkyl group containing 1 to 4 carbon atoms; preferably, it is a linear saturated fatty acid containing 2 carbon atoms;
R3is one of the following structures:
n ranges from 0 to 4.
The compound shown in the formula (I) is an antibacterial agent containing a condensation compound derived from fatty acid and esterified dibasic amino acid, can prevent and treat riemerella anatipestifer disease caused by riemerella anatipestifer, can treat mouse peritonitis diseases caused by escherichia coli and staphylococcus aureus, and can prevent and treat fish septicemia caused by aeromonas sobria. Preferably, the antibacterial agent shown in the formula (I) is lauroyl arginine ethyl ester LAE. The antibacterial agent can effectively inhibit or kill bacteria, greatly reduce the risk of infecting the bacteria of poultry, livestock and aquatic animals, and improve the survival rate of the animals infected with pathogenic bacteria. The lauroyl arginine ethyl ester can be safely degraded in human bodies and animal bodies, has small toxic and side effects, avoids the generation of environment drug-resistant bacteria caused by the discharge of residual antibacterial agents into the environment, achieves effective antibacterial effect and has small negative influence on the environment.
Wherein the prepared medicament is administered at a dose of 0.625-50mg (i.e., 0.625-50 mg/kg bw) of the compound represented by formula (I) per kg body weight (preferably 0.625-45mg/kg bw, more preferably 0.625-40mg/kg bw). In a particular embodiment, the medicament is administered in a dose that gives 2-25mg, 2.5-25mg, 4-25mg, 8-16mg, 10-25mg or 16-25mg, 16-20mg, 30-50mg of the compound of formula (I) per kilogram of body weight, said pathogenic microorganism being a pathogenic gram-positive bacterium, a gram-negative bacterium.
In a preferred embodiment, X is Cl and the compound of formula (I) is lauroyl arginine ethyl ester hydrochloride (LAEHCl) having the formula (II):
in one embodiment, the medicament is an oral medicament for treating or preventing duckling infection with pathogenic gram-negative bacteria, and the medicament is administered in a dose of 8-40mg of the compound represented by formula (I) or (II) per kilogram of body weight. In a preferred embodiment, the medicament is administered in a dose such that 8 to 16mg of the compound of formula (I) or (II) is administered per kilogram of body weight, and the pathogenic gram-negative bacteria are Riemerella anatipestifer, Escherichia coli, Pasteurella, Salmonella, Haemophilus, Brucella. In a most preferred embodiment, the medicament is administered at a dose of 16mg of the compound of formula (I) or (II) per kilogram of body weight, and the pathogenic gram-negative bacterium is Riemerella anatipestifer.
In another embodiment, the medicament is a medicament for intraperitoneal administration for the treatment or prevention of gram-negative bacterial diseases in livestock, aquatic animals, and is administered in a dose of 2.5 to 25mg of the compound represented by formula (I) or (II) per kilogram of body weight. In a preferred embodiment, the pathogenic gram-negative bacteria are riemerella, escherichia coli, pasteurella, salmonella, haemophilus, brucella.
In other embodiments, the medicament is an orally administered medicament for treating or preventing gram positive bacterial diseases in livestock, aquatic animals and is administered in a dose of 2.5-25mg of the compound of formula (I) or (II) per kilogram body weight. In a preferred embodiment, the gram-positive bacterium is staphylococcus aureus, streptococcus, erysipelas, mycobacterium, bacillus anthracis, and the medicament is administered in a dose giving 10-25mg of the compound of formula (I) per kilogram body weight.
In other embodiments, the medicament is an orally administered medicament for treating or preventing gram-negative bacterial diseases in livestock, aquatic animals and the medicament is administered in a dose of 0.625-10mg of the compound represented by formula (I) or (II) per kg body weight. In a preferred embodiment, the gram-negative bacterium is Escherichia coli, Pasteurella, Riemerella, Salmonella, Haemophilus, Brucella, and the medicament is administered in a dose of 0.625-10mg of the compound represented by formula (I) per kg of body weight.
In other embodiments, the medicament is a medicament for treating diarrhea in piglets, and the medicament is administered to the piglets at a dose of 10-50mg of the compound represented by formula (I) or (II) per kg of body weight. In a preferred embodiment, said medicament is administered to the piglets at a dose of 30-50mg of the compound of formula (I) per kg of body weight and achieves a better effect than the same dose of antibiotic in the treatment of diarrhea in piglets. In a most preferred embodiment, the medicament is administered to piglets at a dose of 50mg of the compound of formula (I) or (II) per kg of body weight and achieves a better effect than the treatment of diarrhea in piglets with the same dose of antibiotic.
In another embodiment, the dosage form of the medicament comprising the compound represented by formula (I) or (II) includes granules, solutions, suspensions, powders, capsules, oils, and ointments.
In any of the above embodiments, the livestock, aquatic animal comprises: chicken, duck, goose, turkey, quail, pigeon, pig, cattle, sheep, horse, camel, cat, and fish, shrimp, crab, etc., and the pathogenic bacteria include, but are not limited to, yellow and white dysentery, asthma, erysipelas, edema, clostridial enteritis, proliferative enteritis, tuberculosis, pasteurellosis, anthrax, salmonellosis.
In a preferred embodiment, the compound of formula (I) or (II) kills pathogenic microorganisms within 30min and does not trigger the emergence of drug-resistant bacteria. In another preferred embodiment, the compound of formula (I) or (II) is capable of stimulating pathogenic microorganisms for 30 consecutive days without causing the emergence of drug-resistant bacteria. In a more preferred embodiment, the compound of formula (I) or (II) is less toxic to normal mammalian cells. Does not induce hemolysis of red blood cells at the minimum bactericidal concentration.
in another embodiment, the compound represented by formula (I) or (II) has no influence on the survival rate of healthy ducklings, has no influence on the weight gain of the healthy ducklings, has no toxicity on organs of the healthy ducklings, and can reduce the level of the inflammatory factors IL-1 β and/or IL-1 β protein in animals raised due to bacterial infection.
The second purpose of the invention is to provide the application of the lauroyl arginine ethyl ester derivative shown as the formula (I) or (II) or the hydrate or the pharmaceutically acceptable salt thereof in preparing the medicament for changing the polarity of the cell membrane of the microorganism.
The invention also provides a lauroyl arginine ethyl ester derivative shown as a formula (I) or (II) or a hydrate or a pharmaceutically acceptable salt thereof, and a method for changing the polarity of a cell membrane of a microorganism, which comprises the following steps:
(1) diluting the pathogenic microorganism solution to OD6000.05, then adding a cell membrane polar dye;
(2) adding a compound solution shown in the formula (I) until the final concentration is 16 mu g/ml, and fully reacting;
(3) measuring the fluorescence value at 670nm by using 622nm wavelength light as exciting light;
(4) the fluorescence value of each sample was measured by flow cytometry, and the degree of depolarization of the bacterial cell membrane was analyzed by calculating the ratio.
In a preferred embodiment, X is Cl and the compound of formula (I) is lauroyl arginine ethyl ester hydrochloride (LAEHCl) having the formula (II):
in one embodiment, the microorganism is a pathogenic gram positive bacterium, gram negative bacterium in livestock, aquatic animals.
In a particular embodiment, the gram-positive bacterium is selected from the group consisting of staphylococcus, streptococcus, erysipelothrix, mycobacterium, bacillus anthracis. In a preferred embodiment, the gram-positive bacterium is staphylococcus aureus.
In another specific embodiment, the gram-negative bacteria are selected from the group consisting of E.coli, Pasteurella, Riemerella, Salmonella, Haemophilus, Brucella. In a preferred embodiment, the gram-negative bacteria are selected from the group consisting of E.coli, Riemerella anatipestifer.
In any of the above embodiments, the cell membrane polar dye is dicc3(5)、DiSC2(3) A fluorescent dye.
The invention has the beneficial effects that:
1. the compound shown in the formula (I) or (II) can completely eliminate gram-positive bacteria and gram-negative bacteria (such as escherichia coli, staphylococcus aureus and riemerella anatipestifer) within 30 min.
2. The compound represented by the formula (I) or (II) of the present invention does not induce a drug-resistant mutation of a pathogenic microorganism by continuous administration for 30 days.
3. The compounds of formula (I) or (II) according to the invention do not induce haemolysis of erythrocytes at minimal bactericidal concentrations.
4. The compound shown in the formula (I) or (II) can improve the survival rate of ducklings infected with riemerella anatipestifer, reduce the risk of the riemerella anatipestifer infected with the ducklings, and has no influence on the survival rate of healthy ducklings.
5. The compound shown in the formula (I) or (II) does not influence the weight gain of the ducklings, but can be used as an energy substance to promote the growth of the ducklings.
6. the compound shown in the formula (I) or (II) can restore the increase of the protein levels of inflammatory factors IL-1 β and TNF- α caused by bacterial infection in animals.
7. Experiments prove that when the compound shown in the formula (I) or (II) is continuously orally administered to animals for 3 months, the body weight of an administration group is remarkably increased relative to a control group, and important organs of the administration group have no remarkable toxicity relative to the control group.
8. Experiments prove that the medicine is stopped for 24 hours after the administration for 3 months, and the drug residue of heart, liver, spleen, lung, kidney, small intestine, stomach and muscle tissues of the animals in the administration group meets the requirement of European Union on the drug residue of the non-specified veterinary drugs.
9. Experiments prove that the compound shown in the formula (I) or (II) can promote the growth of poultry, wherein the growth of ducks is promoted, and the daily gain is increased by 11.5%.
10. Experiments prove that the compound shown in the formula (I) or (II) can prevent and treat bacterial diseases of aquatic animals and can promote the vegetative growth of poultry and aquatic animals, wherein: anti-infection experimental results show that all test fishes fed with the feed which does not contain the compound shown in the formula (I) or (II) die, and the survival rate of tilapia fed with the compound shown in the formula (I) or (II) can reach 88.33%; for tilapia, the relative weight gain rate can be improved by 3.03%, 6.23%, 3.88% and the specific growth rate can be improved by 12.32%, and the feed coefficient can be reduced by 3.15%.
11. Experiments prove that compared with the same dosage of gentamicin, the compound shown in the formula (I) or (II) can more effectively treat diarrhea of piglets, avoid the use of antibiotics and reduce the residue of the antibiotics in livestock.
12. The lauroyl arginine ethyl ester is safe to degrade in human bodies and animal bodies, has small toxic and side effects, can avoid the generation of environment-resistant bacteria caused by the emission of residual antibacterial agents to the environment, and has small negative influence on the environment while achieving effective antibacterial effect.
Drawings
FIG. 1(A) shows the time sterilization curve of the lauroyl arginine ethyl ester compound of formula (I) on the clinical isolated pathogenic strain RA-11 of riemerella anatipestifer; (B) the time bactericidal curve of the lauroyl arginine ethyl ester compound of the formula (I) against Staphylococcus aureus ATCC 29213; (C) the time sterilization curve of the lauroyl arginine ethyl ester compound of formula (I) shown against E.coli ATCC 25922.
FIG. 2(A) shows the results of inducing Riemerella anatipestifer drug resistance by using lauroyl arginine ethyl ester compound of formula (I) and chloramphenicol; (B) the result that the lauroyl arginine ethyl ester compound shown as the formula (I) and chloramphenicol induce the staphylococcus aureus drug resistance; (C) the lauroyl arginine ethyl ester compound shown in the formula (I) and chloramphenicol induce the drug resistance of escherichia coli.
The lauroyl arginine ethyl ester compound of formula (I) shown in figure 3 is toxic to mammalian cells. Wherein, the lauroyl arginine ethyl ester compound shown in the (A) has cytotoxicity to mouse fibroblast, mouse myofibroblast and human fibroblast; (B) hemolytic experiment of lauroyl arginine ethyl ester compound on mammalian erythrocytes is shown.
FIG. 4 shows the toxicity results of the lauroyl arginine ethyl ester compound of the formula (I) in animals. Wherein (A) is the survival condition of the duckling orally taking the lauroyl arginine ethyl ester compound for 5 days; (B) the influence of the lauroyl arginine ethyl ester compound on the weight gain rate of the ducklings is shown; (C) orally taking lauroyl arginine ethyl ester compound for mice to obtain the residual amount of the drug in vivo for three months; (D) the change of the body weight of the mouse orally taking the lauroyl arginine ethyl ester compound for three months; (E) orally administering lauroyl arginine ethyl ester compound to mice for three months as main organ index; (F) the result of H & E staining of the 5-day viscera of the duckling orally taking the lauroyl arginine ethyl ester compound; (G) is the H & E staining result of the three-month organs of the mice orally taking the lauroyl arginine ethyl ester compound.
FIG. 5 shows the effect of lauroyl arginine ethyl ester compound of formula (I) on bacterial cell membranes. Wherein (A) is a scanning electron micrograph of normal enterobacter coli; (B) is a scanning electron microscope picture of escherichia coli after the lauroyl arginine ethyl ester compound is treated for 15 min; (C) is a fluorescence spectrophotometer result of the influence of the lauroyl arginine ethyl ester compound on the polarity of the cell membrane of the Riemerella anatipestifer; (D) is a fluorescence spectrophotometer result of the influence of the lauroyl arginine ethyl ester compound on the polarity of staphylococcus aureus cell membranes; (E) is a fluorescence spectrophotometer result of the influence of the lauroyl arginine ethyl ester compound on the polarity of the cell membrane of escherichia coli; (F) is the influence of lauroyl arginine ethyl ester compound on the polarity of red blood cell membrane of mammal; (G) the flow result of the influence of the lauroyl arginine ethyl ester compound on the polarity of the cell membrane of the Riemerella anatipestifer is shown, and the (J) is a statistical chart; (H) the flow result of the influence of the lauroyl arginine ethyl ester compound on the polarity of a staphylococcus aureus cell membrane is shown, and the (K) is a statistical chart; (I) is a flow result of the influence of the lauroyl arginine ethyl ester compound on the polarity of the cell membrane of escherichia coli; (L) is a statistical chart thereof.
The lauroyl arginine ethyl ester compound of formula (I) shown in fig. 6 exerts bactericidal action by binding phosphatidylserine. Wherein (A) is the ITC result of the titration of the lauroyl arginine ethyl ester compound and the aqueous solution; (B) is the titration ITC result of the lauroyl arginine ethyl ester compound and the phosphatidylserine aqueous solution; (C) is the titration ITC result of the lauroyl arginine ethyl ester compound and the phosphatidylcholine aqueous solution; (D) is the titration ITC result of the lauroyl arginine ethyl ester compound and the phosphatidylethanolamine aqueous solution; (E) results of protection of Escherichia coli from lauroyl arginine ethyl ester compounds by phosphatidylserine of different concentrations, and (G) is a statistical chart thereof; (F) results of protection of staphylococcus aureus against lauroyl arginine ethyl ester compound for various concentrations of phosphatidylserine, (H) is its statistical graph; (I) respectively protecting escherichia coli from lauroyl arginine ethyl ester compounds by phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine with the same concentration, wherein (K) is a statistical graph; (J) results of the same concentrations of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine for respectively protecting escherichia coli from lauroyl arginine ethyl ester compounds are shown, and L is a statistical chart thereof.
Detailed Description
The present invention will be described in further detail with reference to the following specific examples and drawings, but the present invention is not limited to the following examples. Variations and advantages that may occur to those skilled in the art may be incorporated into the invention without departing from the spirit and scope of the inventive concept, and the scope of the appended claims is intended to be protected. The procedures, conditions, reagents, experimental methods and the like for carrying out the present invention are general knowledge and common general knowledge in the art, except for the contents specifically mentioned below, and the present invention is not particularly limited.
The first embodiment is as follows: determination of LAE Minimum Inhibitory Concentration (MIC) against bacteria by broth dilution method
The principle and the purpose are as follows: according to the microbubult dilution method specified by CLSI, the minimum drug concentration at which bacterial growth is inhibited after 24h of co-incubation of the drug with bacteria in a 96-well plate is the minimum inhibitory concentration of the drug.
The method comprises the following steps: LAE, bacteria (Riemerella anatipestifer clinical isolates pathogenic strain RA-11, Staphylococcus aureus ATCC29213, and Escherichia coli ATCC 25922) were diluted with Trypticase Soy Broth (TSB) and added to a 96-well plate, respectively, with a blank CK1 medium without bacteria and a CK2 medium supplemented with LAE (1000. mu.g/ml) and a CK3 control medium without drug LAE but containing bacteria that normally grow. The absorbance at 625nm of each well was measured after incubating the 96-well plate in a 37 ℃ incubator for 24 hours. OD of CK1 as compared with blank medium625Wells with consistent values were considered to have no significant growth of bacteria. The lowest concentration of the drug without significant growth of bacteria is the minimum inhibitory concentration MIC (minimum inhibitory concentration) of LAE to bacteriathe run). Adding the liquid in each well onto Tryptone Soy Agar (TSA) medium by stepwise dilution method, evaporating the liquid to dryness, placing in an incubator at 37 deg.C, culturing for 18-24 h, and observing colony formation. The lowest concentration of the drug without colony formation is the minimum bactericidal concentration of LAE to bacteria MBC (minimum bacterial concentration).
As shown in Table 1 below, the minimum bactericidal concentration and the minimum inhibitory concentration of LAE against RA-11 were both not less than 16. mu.g/ml, the minimum bactericidal concentration and the minimum inhibitory concentration against ATCC 25922 were both not less than 16. mu.g/ml, and the minimum bactericidal concentration and the minimum inhibitory concentration against ATCC29213 were both not less than 16. mu.g/ml.
TABLE 1 in vitro antibacterial Effect of LAE on three bacteria
Note: + is turbid bacteria liquid with suspended matter; + is that the bacterial liquid is relatively turbid; the bacteria liquid is clear; for the bacterial liquid to be clear
Example two: determination of effective bacterial Sterilization time of LAE
Principle and purpose: and (3) co-incubating the medicine and the bacteria, taking out a part of samples at regular intervals to count the bacteria flat plates, and thus obtaining a growth curve of the bacteria under the action of the medicine, namely a time sterilization curve of the medicine.
The method comprises the following steps: bacteria in log phase are mixed with LAE solution with different concentrations, and after being added with drugs, the bacteria are sampled for 0, 0.5, 1, 1.5 and 2 hours, and are diluted by 10 times step by step and are placed on a TSA plate to be cultured for 18 to 24 hours at 37 ℃. The bacterial concentration of each drug adding group at each time point is calculated through the colony number, and a bacterial survival curve chart shown in figure 1 is obtained.
And (4) analyzing results: under the concentration of 16 mu g/ml, LAE can completely kill Riemerella anatipestifer and Escherichia coli within 30min, and the quantity of staphylococcus aureus is reduced by 6 orders of magnitude; within 30min, at the concentration of 32 mug/ml, the riemerella anatipestifer, staphylococcus aureus and escherichia coli can be completely killed. Thus, LAE is an antibacterial compound that can kill bacteria quickly, and its short duration of action has the advantage of: can be eliminated before the bacteria generates drug-resistant mutation, and reduces the risk of inducing the bacteria to generate drug resistance.
Example three: LAE-induced bacterial drug resistance assay
The principle and the purpose are as follows: the bacteria can be induced to generate drug-resistant bacteria for the drug under the sub-inhibitory concentration, and the ability of the drug to induce the drug-resistant mutation of the bacteria is detected by transferring the bacteria cultured by the drug with the sub-inhibitory concentration into a fresh drug-containing culture medium for a long time.
The method comprises the following steps: the log phase bacteria were mixed with LAE solution in a glass tube, sealed with a cotton plug, and incubated on a shaker at 37 ℃ and 220rpm for 24 h. Thereafter, the MIC of the drug was checked every 24 hours, and the bacterial solution having the highest drug concentration (i.e., the highest drug concentration lower than the MIC) capable of culturing the turbid bacterial solution was added again to a fresh medium containing different drug concentrations at a ratio of 1:100, and cultured at 37 ℃ and 220rpm for 24 hours for 30 days. Chloramphenicol was used as a positive control group and the procedure was the same as for the LAE group. A statistical map as shown in figure 2 is derived.
And (4) analyzing results:
as shown in fig. 2(a), for riemerella anatipestifer, the MIC of chloramphenicol was first on a continuing ascending trend, rising up to 6-fold of the initial MIC, and then fluctuated, but was substantially higher than the initial MIC, with 30 days of continuous dosing stimulation. The MIC of LAE is basically stable, 1.5 times of the initial MIC appears in one day, and the rest time is lower than or equal to the initial MIC;
as shown in fig. 2(B), with regard to staphylococcus aureus, the MIC of chloramphenicol rapidly rose from day 18 to 10-fold of the initial MIC after 30 days of continuous administration stimulation, and finally stabilized at 8-fold of the initial MIC. . The MIC of LAE is only slightly improved and finally reaches 2 times of the initial MIC;
for E.coli, as shown in FIG. 2(C), after 30 days of continuous administration stimulation, the MIC of chloramphenicol rapidly increased from day 16 and finally reached 12 times the initial MIC. While the MIC of LAE increased only slightly, eventually reaching 2 times the initial MIC. The result shows that the chloramphenicol has higher drug resistance to the riemerella anatipestifer, escherichia coli and staphylococcus aureus, the LAE does not basically induce the drug resistance of the riemerella anatipestifer, and the chloramphenicol has slight drug resistance induction effect on the escherichia coli and the staphylococcus aureus. This property of LAE suggests that it can be an antimicrobial superior to existing antibiotics.
Example four: study of LAE toxicity to mammalian cells in vitro
LAE cytotoxicity assay on mammalian cells:
principle and purpose: MTS is a tetrazolium blue compound, can be reduced into a colored formazan product by dehydrogenase in living cells, and is converted into the survival rate of cells in a sample by measuring the light absorption value of the sample by an enzyme-labeling instrument.
The method comprises the following steps: cytotoxicity experiments: inoculating cells in logarithmic growth phase into 96-well plate at 5000/well amount, changing culture medium into fresh culture medium containing different LAE concentrations after cells adhere to the wall, treating at 37 deg.C for 72h, adding 20 μ l MTS into each well, incubating at 37 deg.C for 1h, measuring absorbance at 490nm of each well with enzyme labeling instrument, and determining OD of control group without administration490Between 0.8 and 1, the data reliability is highest. The cell viability per well was calculated and a graph as shown in fig. 3(a) was prepared. LAE hemolytic test on rabbit erythrocytes:
principle and purpose: after the red blood cells are hemolyzed, contents including colored substances such as hemoglobin and the like are released, cell debris is settled after centrifugation, the colored substances are remained in the supernatant, and the degree of hemolysis of the cells can be converted by detecting the light absorption value of the supernatant.
The method comprises the following steps: collecting fresh rabbit whole blood by ear artery blood sampling method, adding heparin for anticoagulation, centrifuging, cleaning, taking packed red blood cells to prepare 2% red blood cell suspension, and adding 100 μ l of red blood cell suspension into 96-well plate. Equal volumes of LAE solutions with different concentrations are added into the experimental group, equal volumes of PBS are added into the blank control group, equal volumes of 1% Triton X-100 are added into the positive control group, and the experimental group is placed in a constant temperature box at 37 ℃ for 1 h. Centrifuging to obtain supernatant, measuring the light absorption value at 450nm by using a microplate reader, and calculating the hemolysis rate according to the following formula:
a statistical chart shown in fig. 3(B) is obtained.
Analysis of results
As shown in FIG. 3(A), all the cells tested showed high survival rate after 72h of drug treatment, and the survival rate of the cells was more than 80% even at the administration concentration far higher than MIC. According to pharmacopoeia regulations, samples with a haemolysis rate of more than 5% have haemolysis.
As shown in FIG. 3(B), the semi-hemolytic concentration of LAE was 2-fold higher than the MIC of bacteria. This experiment shows that under in vitro conditions, LAE has very low toxicity to mammalian cells and very good selectivity to bacteria.
Example five: controlling effect of LAE on bacterial diseases of livestock
Oral treatment effect of LAE on ducklings infected with Riemerella anatipestifer
The principle and the purpose are as follows: after ducklings are infected with bacteria, the survival condition of animals is observed by orally taking LAE, and the treatment effect of the LAE on poultry with bacterial diseases is studied.
The method comprises the following steps: the ducklings 25 were randomly divided into 5 groups of 5 ducks. Mixing Riemer's rodsThe bacteria count is 4 × 106The CFU amount was subcutaneously injected and inoculated in the legs of four ducklings to be an infected group, and the other group was set as a blank group without any treatment. After 12h, 4 infection groups are respectively administrated with LAE aqueous solution or blank aqueous solution control groups with different dosages in a stomach-irrigation mode, death conditions are observed and recorded at 1h, 7h, 12h, 24h, 48h, 72h and 96h after administration, and the survival conditions of 96h animals are shown in a table 2.
Table 2: therapeutic effects of oral LAE on ducklings infected with Riemerella anatipestifer
Note: the test group was animals infected with pathogenic bacteria and given varying concentrations of LAE;
the blank group is animals which are not contacted with pathogenic bacteria;
the control group was animals infected with pathogens administered only with the blank aqueous solution but not with the LAE aqueous solution.
And (4) analyzing results: after the ducklings are infected with riemerella anatipestifer, if the ducklings are not treated by the medicine, the ducklings die in 48 hours; if LAE aqueous solution is used for intragastric administration treatment 12h after infection, the survival rate of 96h after infection reaches 60%, and the oral dosage of LAE at least 8-16 mg/kg bw can effectively reduce the morbidity of the duckling Duemerella anatipestifer duckling by considering the treatment period of 4-8 days and the cost of LAE treatment solvent, thereby meeting the production requirement. Thus, LAE greatly improves the survival rate of infected ducklings.
Control effect of LAE on riemerella anatipestifer infection of ducklings
The principle and the purpose are as follows: before bacterial infection, the ducklings take LAE orally, after infection, the LAE continues to take orally every day, the survival condition of animals is observed, and the effect of the LAE serving as an antibacterial agent for protecting poultry from bacterial infection is studied.
The method comprises the following steps: the ducklings 50 were randomly divided into 5 groups of 10 ducks. Wherein 2 groups were administered in the form of gavagesBlank aqueous solution was used as the non-administered group, and 3 groups were used as the test groups by gavage with an aqueous LAE solution. The Riemerella anatipestifer is administered at 4 × 10 for 8h after the first administration6The legs of ducklings inoculated in three experimental groups and one non-administration control group were used as infected groups by subcutaneous injection of the amount of CFU, and the other 1 non-administration group was used as a non-infected group without any treatment (i.e. without infection of bacteria and administration as blank). The blank aqueous solution and the infected group were administered in intragastric administration at 12h, 24h and 48h after infection, respectively, and the experiment 1, 2 and 3 groups were administered with LAE aqueous solutions of different concentrations, respectively. Death was observed and recorded at 1h, 7h, 12h, 24h, 48h, 72h, 96h post-dose, and the results for 96h survival are shown in table 3.
Table 3: the oral LAE has the effect of preventing and treating duck plague Lymerella anatipestifer infection.
Note: the test group comprises animals infected with pathogenic bacteria after being given LAE with different concentrations;
blank group is animals not exposed to pathogenic bacteria and not administered LAE;
the control group was animals infected with pathogenic bacteria but not administered LAE.
And (4) analyzing results: oral administration of LAE before infection of ducklings with bacteria can improve the survival rate of ducklings again compared with administration after infection. The oral dosage of LAE of 8-40 mg/kg bw can effectively prevent and treat duck infection of Riemerella anatipestifer. The continuous administration can exert the effect of preventing and treating diseases to a greater extent. All the 10 ducklings in the solvent control group die within 96h, and 9 ducklings in the administration group survive at the end of the experiment, and the survival rate reaches 90%. Considering the 4-8 day treatment cycle and the cost of LAE therapeutic solvent, a dosage range of 40mg/kg bw has been the upper limit of the production requirement.
Study of the therapeutic Effect of intraperitoneal administration of LAE on mice infected with Escherichia coli
The principle and the purpose are as follows: injecting colibacillus into the abdominal cavity of a mouse to simulate the livestock to be infected with gram negative bacteria diseases, carrying out abdominal administration after infection to observe the survival rate of the mouse, detecting the clearance rate of LAE on pathogenic bacteria in the mouse, and detecting the influence of a medicament on the level of inflammatory factors in the infected mouse. The experiment can detect the treatment effect of LAE on livestock infected with gram-negative bacteria.
The method comprises the following steps: 50 Balb/c mice were randomly divided into 5 groups of 10 mice each. The log phase of E.coli was scaled to 108The amount of CFU was injected into the abdominal cavity of 4 groups of mice, and the other group was set as an uninfected group without any treatment. After 1h, 1 infected group was injected with 0.5ml of physiological saline intraperitoneally, and the other three infected groups were injected with LAE solutions of different concentrations, respectively. Mice survived 24h after statistics, the results of table 4 were obtained.
An additional 60 Balb/c mice were randomly assigned to 6 groups of 10 mice each. The log phase of E.coli was scaled to 108injecting the amount of CFU into the abdominal cavity of 5 groups of mice, setting the other group as an uninfected group without any treatment, injecting 0.5ml of physiological saline into the abdominal cavity of 1 infected group after 1 hour, respectively injecting LAE solutions with different concentrations into the other 4 infected groups, killing the mice by using a cervical dislocation method after administration for 24 hours, flushing the abdominal cavity of the mice by using sterile PBS, collecting the abdominal cavity fluid, spotting the abdominal cavity fluid on a TSA plate by using a stepwise dilution method, inversely culturing for 18-24 hours at 37 ℃, counting and calculating the concentration of pathogenic bacteria in the abdominal cavity fluid of each group of mice, respectively detecting the levels of IL-1 β and TNF- α in the abdominal cavity fluid by using ELISA kits of IL-1 β and TNF- α, and the results are shown in Table 5.
Table 4: therapeutic effect of intraperitoneal administration of LAE on mammals infected with E.coli
Note:
the test group is animals infected with pathogenic bacteria and orally administered with LAE of different concentrations;
the blank group is animals which are not contacted with pathogenic bacteria;
the control group was a group infected with pathogenic bacteria but not administered LAE.
Table 5: effect of intraperitoneal LAE on eliminating pathogenic bacteria in mammals infected with escherichia coli
Note:
the test group was animals infected with pathogenic bacteria and given varying concentrations of LAE;
the blank group is animals which are not contacted with pathogenic bacteria;
the control group was animals infected with pathogenic bacteria but not administered LAE.
And (4) analyzing results: after the mice are infected with escherichia coli, 10 animals injected with the normal saline group die within 11h, and the survival rate of the mice injected with the LAE solution is 70% after 24h, which shows that the LAE has a therapeutic effect on the animals, eliminates pathogenic escherichia coli in the mice and reduces the level of inflammatory factors raised due to bacterial infection.
The survival rate of mice infected with escherichia coli can be improved by intraperitoneal injection of 2.5-25mg of LAE per kg of body weight, and pathogenic bacteria in the infected mice can be eliminated by intraperitoneal injection of 1-25 mg of LAE per kg of body weight.
The treatment effect of oral LAE administration on mice infected with gram-negative bacteria and gram-positive bacteria.
The principle and the purpose are as follows: the oral ingestion and the exertion of the curative effect are high requirements for antibacterial agents, so that mice are used for representing livestock, the mice are infected by gram-negative bacteria escherichia coli and gram-positive bacteria staphylococcus aureus respectively, and then the treatment effect of the medicine on animals is evaluated by orally taking LAE.
The method comprises the following steps: 60 Balb/c mice were randomly divided into 6 groups of 10 mice each. The log phase of Staphylococcus aureus was treated with 108The amount of CFU was injected into the abdominal cavity of the mice. And (3) respectively irrigating 0.5ml of physiological saline, LAE aqueous solutions with different drug concentrations or cefazolin aqueous solutions after 1h, killing the mice by a cervical dislocation method after administration for 24h, flushing the abdominal cavity of the mice with sterile PBS, and collecting the abdominal cavity liquid. The peritoneal fluid was spotted on a TSA plate by stepwise dilution and cultured in an inverted state at 37 ℃ for 18-24 h. The concentration of pathogenic bacteria in the peritoneal fluid of each group of mice was counted and calculated, and the results are shown in table 6.
Table 6: scavenging effect of oral LAE against gram-positive pathogenic bacteria in mammals
Note:
the test group is animals infected with pathogenic bacteria and orally administered with LAE with different concentrations;
the cefazolin group is animals infected with pathogenic bacteria and orally taking cefazolin aqueous solution;
the blank group is animals which are not contacted with pathogenic bacteria;
the control group was animals infected with pathogenic bacteria but not administered.
30 Balb/c mice were randomly divided into 6 groups of 5 mice each. The log phase of E.coli was scaled to 108The amount of CFU was injected into the abdominal cavity of the mice. After 1h, 5 groups of the medicinal liquid are orally administrated with 0.5ml of normal saline, LAE aqueous solution with different concentrations and ampicillin aqueous solution respectivelyMice were sacrificed 24h post-dose cervical dislocation, the abdominal cavity of the mice was rinsed with sterile PBS and the abdominal cavity fluid was collected. The peritoneal fluid was spotted on a TSA plate by stepwise dilution and cultured in an inverted state at 37 ℃ for 18-24 h. The concentration of pathogenic bacteria in the peritoneal fluid of each group of mice was counted and calculated, and the results are shown in Table 7.
Table 7: scavenging effect of oral LAE against gram-negative pathogenic bacteria in mammals
Note:
the test group is animals infected with pathogenic bacteria and orally administered with LAE with different concentrations;
the ampicillin mycin group is animals infected with pathogenic bacteria and orally taking ampicillin aqueous solution;
the blank group is animals which are not contacted with pathogenic bacteria;
the control group was animals infected with pathogenic bacteria but not administered.
And (4) analyzing results: the oral administration of LAE after the mice are infected with bacteria can reduce the amount of escherichia coli and staphylococcus aureus in the infected mice, and the oral administration of 2.5-25 milligrams of LAE per kilogram of body weight can eliminate pathogenic gram positive bacteria staphylococcus aureus in the sick mice; oral administration of 0.625-10mg per kg body weight of LAE eliminates the pathogenic gram negative bacteria escherichia coli in diseased mice. The scavenging effect is equivalent to the curative effect of antibiotics, which indicates that LAE can be used for treating gram-negative bacteria and gram-positive bacteria diseases of mammals in an oral administration mode.
In conclusion, LAE can be used for oral treatment and prevention of bacterial diseases in poultry; the LAE can be used for treating gram-positive bacteria and gram-negative bacteria infected livestock by oral administration; the LAE can be administered intraperitoneally to treat bacterial infections in livestock.
Example six: toxicity study of LAE on livestock
Toxicity study of LAE oral administration on cherry duck ducklings
The principle and the purpose are as follows: once a day, LAE with four times of effective treatment concentration is orally taken for one week, the mental state, survival rate and weight increase of animals are observed, and organ toxicity is detected.
The method comprises the following steps: the ducklings 20 were randomly divided into 2 groups of 10 ducks. Wherein, the group A is administered with a blank aqueous solution in a gastric lavage manner, the group B is administered with LAE aqueous solution with four times of effective treatment concentration in a gastric lavage manner, the drug is administered once every 24 hours, the administration is continuously carried out for five days, 7 days are continuously observed, the clinical manifestations of the experimental animals are recorded to obtain a survival rate statistical chart shown in figure 4(A), and the heart, liver, spleen and kidney tissues of the experimental animals in the blank group and the group with the highest drug dose are taken to carry out pathological analysis at the end of the experiment. The results of organ toxicity shown in FIG. 4(F) were obtained. And the average daily gain of the experimental animals in each group was counted for 7 days as shown in FIG. 4 (B).
Study of acute and subacute toxicity and in vivo accumulation of LAE by oral administration
The principle and the purpose are as follows: the safety of LAE was evaluated by studying the long-term toxicity of oral LAE to animals and the accumulation of drugs in vivo. Study of drug accumulation in animals analyzed whether LAE could be used as a residue-free additive.
The method comprises the following steps: duckling acute toxicity test: 20 ducklings are randomly divided into two groups, and the gavage is performed every day, wherein the experimental group is LAE aqueous solution, and the blank control group is water with the same volume. Orally taking for 7 days, measuring body weight, and H & E staining heart, liver, spleen, lung and kidney; mouse subacute toxicity test: 20 mice were randomly divided into two groups of 10 mice each. Mice were given daily by gavage with the experimental group containing LAE water solution and the blank control group containing the same volume of water. The mice were weighed weekly, sacrificed at 24H after the last dose after 90 days, weighed heart, liver, spleen, lung, kidney, H & E stained and tested for the residual amount of LAE in each tissue and organ by HPLC.
And (4) analyzing results:
(1) duckling acute toxicity test: none of the experimental animals died, and all the groups administered showed no abnormality in morphology, mental state, hair color, etc. from the control group.
(2) The appearance of the organs is not abnormal, and H & E staining of heart, liver, spleen and kidney tissue sections does not show abnormality with the control group. The average daily gain of the blank control group is 21.38g, the average daily gain of the highest drug dosage group is 22.57g, and the drug does not cause negative influence on the weight gain of the duck. Under the dosage of the medicament which is four times of the medicament for effectively preventing and treating riemerella anatipestifer, the LAE has no oral toxicity to the cherry duck ducklings;
(3) mouse subacute toxicity test: the mice in the experimental group showed significant increases in body weight gain over the blank group at week 1 and weeks 4 to 10 over 90 days of oral administration as shown in fig. 4 (D). The graph E shows that the LAE has no obvious influence on organ coefficients, and the H & E staining result of each organ of the graph G shows that the LAE has no obvious toxicity on organs of experimental animals. The LAE content in each tissue and organ (FIG. C) was less than 10. mu.g/kg (the European Union requires no veterinary drug).
The above results show that oral administration of LAE to mammals for 3 months has very low toxicity to animals, can promote weight gain of animals to some extent, and has very low drug residue. The results show that the LAE has extremely low acute and subacute toxicity to animals, has extremely low accumulation in vivo after long-term administration, and meets the requirements of safety and environmental protection of antibacterial agents.
Example seven: LAE compounds as feed additives for the prevention of diseases in mammals
Directly adding the active ingredients of the LAE compound into a mouse feed in an amount of 0.01-1.0% by weight of the total weight of the feed; or mixing the product with carrier to obtain premix; or mixing with other feed additives or feed raw materials to make into premix and concentrated feed for feeding mice.
50 mice of 6 weeks old were selected and randomly divided into 5 treatment groups according to the principle of similar body weight. The control group is fed with corn-wheat-soybean-meal type daily ration, the test group is fed with corn-wheat-soybean-meal type daily ration, 0.01 percent, 0.1 percent and 1 percent of LAE compound by total weight of the feed are added, and after the control group is fed for 7 days, the disease resistance of the mice to escherichia coli is measured. The results are shown in Table 8.
Table 8: disease control in mammals with feed-supplemented LAE
Note:
test groups were feeds exposed to pathogens and supplemented with varying amounts of LAE compound;
the blank group was not exposed to pathogenic bacteria;
the control group was a feed exposed to pathogenic bacteria without addition of LAE compound
As can be seen from the above table, the survival rates of mice in all the test groups are significantly increased compared with the control group, wherein the best group is the test 3 group, the survival rate is 80%, and all the experimental animals in the control group without addition of LAE die, which indicates that the LAE compound has a prevention and treatment effect on the diseases of mammals as a feed additive. Considering the treatment period and the cost of the LAE therapeutic agent, the effective concentration of the LAE as a medicament or feed additive is 0.01-1%, which can effectively prevent and treat the diseases and meets the production requirement.
EXAMPLE VIII LAE Compounds as nutritional energy supplements for the vegetative growth of mammals
According to the application range of the concentration of the LAE compound determined in the seventh embodiment, on the basis of the same daily ration formula of each group, the LAE compound is added into the complete formula feed by 0.3%, 0.6% and 0.9% in mass ratio respectively in the groups of experiments 1, 2 and 3, and no drug is added into the control group. 40 Balb/c mice of 6 weeks old were randomly divided into 4 groups, and the weight of each group was weighed at day 1 at the start of the test and again at day 15 later. The results are shown in Table 9:
table 9: effect of feed additive LAE on mammalian growth
Note:
the test groups were diets supplemented with different doses of LAE compound;
the control group was a feed without addition of LAE compound
And (4) analyzing results: as can be seen from the above table, the differences between the initial weight of the test and the groups are not significant. From the individual net weight gain on 15 scales, 2 groups were significantly higher than 1 and 3 groups.
Therefore, compared with a control group, the addition of 0.6% of LAE compound in the mouse feed during the test period obviously improves the average weight gain of the mouse by 9.64%. Thus, the addition of the LAE compound to the feed can promote the growth of the mammal.
Example nine: LAE compound as feed additive for preventing and treating diseases of ducklings
The effective components of the LAE compound account for 0.01-1.0 percent of the total weight of the feed, and the LAE compound is directly added into duckling feed; or mixing the product with carrier to obtain premix; or mixing with other feed additives or feed raw materials to prepare a premix and a concentrated feed for feeding ducklings.
50 cherry ducklings of 14 days old are selected and randomly divided into 5 treatment groups according to the principle of similar weight. Each treatment was repeated 1 time (column), and each column was 10 (male and female halves). The control group is fed with corn bean pulp type daily ration, the test group is fed with corn bean pulp type and added with LAE compound accounting for 0.01 percent, 0.1 percent and 1 percent of the total weight of the feed, and after the ducklings are fed for 7 days, the disease resistance of the ducklings to riemerella anatipestifer disease is measured. The results are shown in Table 10.
Table 10: the disease control effect of the feed added with LAE on ducklings is as follows:
note:
the test groups were diets supplemented with different doses of LAE compound;
the blank group was not exposed to pathogenic bacteria;
the control group was a feed without addition of LAE compound
And (4) analyzing results: as can be seen from the above table, the survival rate of the ducklings in all test groups is significantly increased compared with that of the control group, wherein the best group is the test group 3, the survival rate is 90%, and all experimental animals in the control group without addition of LAE die, which indicates that the LAE compound has a prevention and treatment effect on ducklings diseases as a feed additive, and the effective concentration of the LAE compound as a medicament or feed additive is 0.01-1%, preferably 0.1-1%, in consideration of the treatment period and the cost of the LAE therapeutic agent, so that the onset of disease can be effectively prevented and treated, and the requirement of production is met.
EXAMPLE ten LAE Compounds as nutritional energy supplements for disease prevention and vegetative growth of ducklings
According to the application range of the concentration of the LAE compound determined in the ninth embodiment, on the basis of the same daily ration formula of each group, the LAE compound is added into the complete formula feed by 0.3%, 0.6% and 0.9% in mass ratio respectively in the groups of experiments 1, 2 and 3, and no drug is added into the control group. Weighing the weight of each group of animals at the 1 st day when the test is started, weighing the weight again after 15 days, and measuring the disease resistance of each group to the Riemerella anatipestifer disease. The results are shown in Table 11:
table 11: disease prevention and treatment and vegetative growth effect of ducklings by adding LAE into feed
Note:
test groups were diets supplemented with varying amounts of LAE compound;
the control group was a feed without addition of LAE compound
And (4) analyzing results: therefore, compared with a control group, the average weight gain of the ducklings can be improved by adding 0.3-0.9% of LAE in the ducklings feed in the test period, wherein the weight gain effect of the 0.6% LAE compound is most obvious, and the average weight gain of the ducklings is obviously improved by 11.5% compared with the control group. Therefore, the LAE compound can reduce the death of the ducklings caused by Riemerella anatipestifer disease, improve the growth performance of the ducklings and promote the vegetative growth of the ducklings.
Example eleven: LAE compounds as nutritional energy supplements for preventing diseases and for the vegetative growth of aquatic animals
240 tilapia with the weight of 42.1 +/-0.24 g is selected to be divided into 4 treatment groups, LAE compounds with different gradient levels are added respectively, each group has 3 repetitions, and each repetition has 20 fishes. The effect of LAE compound on the growth performance of tilapia was evaluated by 8 weeks of growth test. When the tilapia is fed with the LAE feed for 8 weeks, aeromonas sobria infection is carried out to explore the capability of the LAE to resist bacterial diseases of aquatic animals, and the disease resistance of the tilapia is reflected by the survival rate of the infected tilapia. The results are shown in Table 12.
TABLE 12 Effect of feed addition of LAE on the vegetative growth of Tilapia mossambica
Note:
the test groups were fed with different doses of LAE;
the control group was a feed without addition of LAE compound
The data in Table 12 show that the addition of LAE compound in tilapia feed can improve the relative weight gain rate by 3.03%, 6.23% and 3.88% and the specific growth rate by 5.69%, 12.32% and 7.1% (P is less than 0.05), reduce the feed coefficient by 2.36%, 3.15% and 1.57% (P is less than 0.05), and promote the growth of tilapia.
Meanwhile, anti-infection experimental results show that all the test fishes eating the feed without LAE die, and the survival rates of the tilapia eaten with LAE are 61.67%, 78.33% and 88.33% respectively. Therefore, the anti-infection capability of the fish can be improved by adding 0.01 to 1 percent of LAE.
Example twelve: LAE triggers bacterial death by depolarizing the bacterial cell membrane
SEM Observation of the Effect of LAE on bacterial morphology
Principle and purpose: the surface appearance of a sample after being fixed, dehydrated and sprayed with gold can be observed under a scanning electron microscope, and the technology is usually used for analyzing the microscopic surface appearance of chemical materials and biological materials.
The method comprises the following steps: coli in log phase was treated with 1 × MIC LAE for 15 min. Centrifuging at 8000rpm for 5min, discarding supernatant, adding 2.5% glutaraldehyde for fixation for 2h, blowing with PBS for three times, 10min each time, fixing with 1% osmic acid for 1h, and blowing with PBS for 10min each time. Sequentially dehydrating with 50%, 70%, and 90% ethanol for 10min, and dehydrating with anhydrous ethanol for 5 min. Drying for 2h by critical point drying method. The dried powder was stuck on a stage and sprayed with gold for 40 min. And observing and taking a picture by using a scanning electron microscope.
2. The influence difference of LAE on the polarity of prokaryotic and eukaryotic cell membranes is researched by dyeing with fluorescent dye.
Principle and purpose: DiSC3(5)、DiSC2(3) The fluorescent dye is a polar dye of cell membrane, and the cell membrane passes through DiSC due to the existence of internal and external potential difference2(3) Emitting red fluorescence after dyeing, generating depolarization reaction once the potential difference disappears, DiSC2(3) Green fluorescence is emitted, and the strength of depolarization reaction of the cells can be obtained by calculating the ratio of the red fluorescence value to the green fluorescence value.
The method comprises the following steps: logarithmic phase RA-11 was diluted to OD with PBS600Adding DiSC 0.05 ═ 0.053(5) The fluorescent dye is placed in the dark at 37 ℃ for 1h, and then KCl solution is added. Adding LAE solutions with different concentrations to make their final concentrations respectively 0.5 × MIC and MIC, and respectively taking polymyxin B, vancomycin and PBS as positive control, negative control and blank control. The light with wavelength of 622nm is used as excitation light, and the fluorescence value at 670nm is measured at 0, 2, 10, 20, 30, 40, 50 and 60min after the dosing, so as to make a statistical chart as shown in FIG. 5 (C).
Log phase Staphylococcus aureus diluted to OD with PBS600When the concentration is 0.05, the DiSC3(5) fluorescent dye is added, and the mixture is kept at 37 ℃ in the dark for 1 hour and then added with KCl solution. Adding LAE solutions with different concentrations to make their final concentrations respectively 0.5 × MIC and MIC, and respectively taking polymyxin B, vancomycin and PBS as positive control, negative control and blank control. The fluorescence at 670nm was measured at 0, 2, 10, 20, 30, 40, 50, and 60min after the application of the laser light with a wavelength of 622nm as the excitation light, and a statistical chart as shown in FIG. 5(D) was prepared.
Logarithmic phase E.coli was diluted to OD with PBS600When the concentration is 0.05, the DiSC3(5) fluorescent dye is added, and the mixture is placed at 37 ℃ in the dark for 1 hour and then KCl solution is added. Respectively adding LAE solution with different concentrations to make final concentrations respectively 0.5 × MIC and MIC, respectively collecting polymyxin B, vancomycin and PBS is a positive control, a negative control and a blank control. The fluorescence value at 670nm was measured at 0, 2, 10, 20, 30, 40, 50, and 60min after the addition of the excitation light at 622nm wavelength, and a statistical chart as shown in FIG. 5(E) was prepared.
Fresh rabbit erythrocytes were counted and diluted to 5X 107Adding DiSC3(5) fluorescent dye into the mixture per ml, standing the mixture at 37 ℃ in the dark for 1 hour, and adding a KCl solution. LAE solutions of different concentrations were added to give final concentrations of 0.5 × MIC and MIC for E.coli, respectively, and light of 622nm wavelength was used as excitation light, and the fluorescence value at 670nm was measured at 0, 2, 10, 20, 30, 40, 50, and 60min after the addition of the drugs, respectively, to give a statistical chart as shown in FIG. 5 (F).
Dilution of Riemerella anatipestifer in log phase to 5X 107CFU/ml, different concentrations of LAE solution were added to give final concentrations of 0.5 × MIC, 2 × MIC, respectively. And the group added with CCCP is taken as a positive control, and the group added with PBS is taken as a negative control. Addition of DiOC2(3) After 1 hour of treatment, the flow cytometer measured the red and green fluorescence values of each sample, and the degree of depolarization of the bacterial cell membrane was analyzed by calculating the ratio as shown in fig. 5(G), and a statistical chart as shown in fig. 5(J) was prepared.
Log phase dilution of Staphylococcus aureus to 5X 107CFU/ml, different concentrations of LAE solution were added to give final concentrations of 0.5 × MIC, 2 × MIC, respectively. And the group added with CCCP is taken as a positive control, and the group added with PBS is taken as a negative control. Addition of DiOC2(3) After 1 hour of treatment, the red and green fluorescence values of each sample were measured by flow cytometry, and the degree of depolarization of the bacterial cell membrane was analyzed by calculating the ratio as shown in FIG. 5(H), and a statistical chart as shown in FIG. 5(K) was prepared.
Logarithmic phase E.coli dilution to 5X 107CFU/ml, different concentrations of LAE solution were added to give final concentrations of 0.5 × MIC, 2 × MIC, respectively. And the group added with CCCP is taken as a positive control, and the group added with PBS is taken as a negative control. Addition of DiOC2(3) After 1 hour of treatment, the red and green fluorescence values of each sample were measured by flow cytometry, and the bacterial cells were analyzed by calculating the ratio as shown in FIG. 5(I)The degree of membrane depolarization was calculated and a statistical map was made as shown in fig. 5 (L). And (4) analyzing results: scanning electron microscope results show that when 1 XMIC LAE is used for treating escherichia coli for 15min, pits or even cavities appear on the surface of thalli, which indicates that LAE can destroy the integrity of bacteria to cause bacterial death. For riemerella anatipestifer and escherichia coli, fluorescence values of all the LAE application groups and the positive control polymyxin B groups can be greatly improved in a short time, which indicates that LAE can enable cell membranes to have depolarization reaction, and fluorescence values of the negative control vancomycin groups and the blank control groups do not obviously change; for staphylococcus aureus, all the LAE application groups and the positive control polymyxin B group can greatly improve the fluorescence value in a short time, which indicates that LAE can enable a cell membrane to generate depolarization reaction, and the fluorescence values of the negative control vancomycin group and the blank control group are not obviously changed; in the case of mammalian erythrocytes, only a slight depolarization occurs when the concentration of LAE reaches 2 times the MIC of E.coli. The experimental result shows that the LAE can break the membrane through depolarization on the bacterial cell membrane so as to play a bactericidal effect, and the effect on prokaryotic cells is stronger than that on eukaryotic cells, which is caused by the phenomena of strong bactericidal effect and low toxicity of the eukaryotic cells. The flow result is consistent with the detection result of the spectrophotometer.
Example thirteen: LAE triggers bacterial death by binding phosphatidylserine on the bacterial cell membrane
1. Isothermal titration calorimetry for detecting LAE binding to phospholipids
Principle and purpose: when two molecules are combined, heat is absorbed or released, and whether the two molecules have the combination reaction can be determined by detecting the change of heat when the two molecules are titrated mutually.
The method comprises the following steps: the aqueous LAE solution was titrated on ITC200 apparatus with Phosphatidylcholine (PC), Phosphatidylethanolamine (PE), and Phosphatidylserine (PS) solutions, respectively, and the respective thermal change curves were recorded and the binding constants of LAE to the respective phospholipid molecules were calculated. A curve like that of FIG. 6A/B/C/D was obtained.
As a result: only the caloric change profile of phosphatidylserine and LAE during titration was matched to the caloric change of the binding of the two molecules, with a binding constant of about 2.95E5 + -2.48E 5M‐1. While phosphatidylcholine and phosphatidylethanolamine did not detect a binding reaction. Indicating that LAE can be combined with acidic phospholipids such as phosphatidylserine on the surface of the bacterial membrane, but not with neutral phospholipids. 2. Phospholipid protection assay
Principle and purpose: according to the above test we can preliminarily judge that there is a binding reaction between LAE and phosphatidylserine molecule, if this binding is a necessary factor for LAE to exert bactericidal function, then when we add a certain amount of PS in the system where LAE and bacteria are incubated together, we will reduce the bactericidal efficacy of LAE, and neither adding PE nor PC will play the same role.
The method comprises the following steps: and low-concentration or high-concentration phosphatidylserine PS is respectively added into gram-negative bacteria escherichia coli and gram-positive bacteria staphylococcus aureus. Then, an aqueous LAE solution of the same concentration was added thereto, and the mixture was cultured at 37 ℃ for 24 hours. Counting viable bacteria on a flat plate of each sample, carrying out inverted culture in an incubator at 37 ℃ for 18-20 hours, and then photographing to obtain the conditions of resisting LAE under the protection of PS for the escherichia coli shown in figure 6E and the staphylococcus aureus shown in figure 6F respectively, wherein figures 6G/H are respective statistical graphs. In addition, we also use the same concentrations of phosphatidylcholine PC, phosphatidylethanolamine PE, phosphatidylserine PS to repeat the above experiments, and get the results of FIG. 6I Escherichia coli, FIG. 6J Staphylococcus aureus resistant to LAE under the protection of phospholipids, respectively, and FIG. 6K/L is its respective statistical chart.
As a result: consistent with expectations, only phosphatidylserine PS was able to protect bacteria from the killing effect of LAE. It was concluded that LAE exerts its bactericidal effect by binding to PS.
EXAMPLE fourteen comparison of the therapeutic Effect of LAE and antibiotics on diarrhea piglets
The applicant entrusts a large pig farm in Henan province to perform SY (LAE of the present invention) administration experiments on 118 piglets with diarrhea.
A gentamicin administration group was also set as a parallel control.
The affected pigs were drenched 3 times a day in the morning (7:00), in the middle (12:00) and at night (18: 00).
Evaluation criteria: animals without drug administration after diarrhea treatment are counted as remaining animals, namely the test is stopped;
the animals that died inefficiently after the piglets were dosed were counted as dead animals, i.e. the test was stopped.
The results are shown in Table 13.
Watch 13
As shown in the table above, the average cure rate for piglets treated with gentamicin was 77.5%. However, this dose has far exceeded the dose used with conventional antibiotics, suggesting that antibiotic residues in piglets will be significantly out of the limits in order to achieve this cure rate.
The cure rate of the piglet treated by the LAE is 72.2% at the minimum dose of 10mg/kg, and 78.57% at the maximum dose of 50mg/kg, which both far exceed the cure rate of the antibiotic at the same dose (the cure rate of gentamicin at 10mg/kg is not shown), which indicates that the cure rate of the LAE at the same dose is higher than that of gentamicin when the LAE is used for treating diarrhea of the piglet, and due to the good biological metabolism characteristic, the LAE indicates that no harmful drug residue exists in the piglet body, and provides a good direction for replacing the antibiotic for animals.
Claims (23)
1. An application of a compound shown as a formula (I) or a hydrate or a pharmaceutically acceptable salt thereof in preparing a medicament for treating or preventing diseases of livestock and aquatic animals caused by pathogenic microorganisms,
wherein X is halogen or HSO4；
R1Is a linear saturated fatty acid radical having from 8 to 14 carbon atoms, orAn oxo acid group having 8 to 14 carbon atoms;
R2is a linear fatty acid group containing 1 to 18 carbon atoms, or a branched fatty acid group containing 1 to 18 carbon atoms, or an aromatic group containing 1 to 18 carbon atoms or a linear alkyl group containing 1 to 4 carbon atoms;
R3is one of the following structures:
n ranges from 0 to 4.
2. The use according to claim 1, wherein X is Cl and the compound of formula (I) is lauroyl arginine ethyl ester hydrochloride having the formula (II):
3. use according to claim 1 or 2, wherein the medicament is to be administered in a dose of 0.625 to 50mg of the compound of formula (I) per kg of body weight.
4. Use according to claim 3, wherein the medicament is administered in a dose that gives 2 to 25mg, 2.5 to 25mg, 4 to 25mg, 8 to 16mg, 10 to 25mg or 16 to 25mg, 16 to 20mg, 30 to 50mg of the compound of formula (I) per kg of body weight, the pathogenic microorganism being a pathogenic gram-positive bacterium, a pathogenic gram-negative bacterium.
5. The use according to claim 3, wherein the medicament is an oral medicament for the treatment or prevention of duckling infection with pathogenic gram-negative bacteria and is administered in a dose of 8-40mg of the compound of formula (I) per kilogram of body weight.
6. Use according to claim 5, characterized in that the medicament is administered in a dose that gives 8 to 16mg of the compound of formula (I) per kilogram of body weight, the pathogenic gram-negative bacteria being Riemerella anatipestifer, Escherichia coli, Pasteurella, Salmonella, Haemophilus, Brucella.
7. The use according to claim 6, wherein the medicament is administered in a dose that gives 16mg of the compound of formula (I) per kilogram of body weight, and the pathogenic gram-negative bacterium is Riemerella anatipestifer.
8. Use according to claim 3, wherein the medicament is to be administered in a dose such that 2.5-25mg of the compound of formula (I) is administered per kilogram of body weight.
9. The use according to claim 8, wherein the pathogenic gram-negative bacteria are riemerella, escherichia coli, pasteurella, salmonella, haemophilus, brucella.
10. Use according to claim 3, wherein the medicament is an orally administered medicament for the treatment or prevention of gram positive bacterial diseases in livestock, poultry, aquatic animals and is administered in a dose of 2.5-25mg of the compound of formula (I) per kg body weight.
11. Use according to claim 10, wherein the gram-positive bacterium is staphylococcus aureus, streptococcus, erysipelothrix, mycobacterium, bacillus anthracis, and the medicament is to be administered in a dose giving 10-25mg of the compound of formula (I) per kilogram of body weight.
12. Use according to claim 3, wherein the medicament is an orally administered medicament for the treatment or prevention of gram-negative bacterial diseases in livestock, poultry, aquatic animals and is administered in a dose of 0.625-10mg of the compound of formula (I) per kg body weight.
13. The use according to claim 12, wherein the gram-negative bacterium is escherichia coli, pasteurella, riemerella, salmonella, haemophilus, brucella, and the medicament is administered in a dose that gives 0.625-10mg of the compound of formula (I) per kg of body weight.
14. Use according to claim 3, wherein the medicament is a medicament for the treatment of diarrhoea in piglets, and which is administered to the piglets at a dose of 10-50mg of a compound of formula (I) or (II) per kg body weight.
15. The use according to claim 14, wherein the medicament is for administration to piglets at a dose of 30-50mg of a compound of formula (I) or formula (II) per kg body weight.
16. The use according to claim 15, wherein the medicament is for administration to piglets at a dose of 50mg of the compound of formula (I) per kg of body weight.
17. The use according to any one of claims 4 to 16, wherein the medicament is in the form of granules, solutions, suspensions, powders, capsules, oils, pastes.
18. The use as claimed in claim 17, wherein said livestock, aquatic animals comprise: chicken, duck, goose, turkey, quail, pigeon, etc., pig, cattle, sheep, horse, camel, cat, dog, fish, shrimp, crab, etc.
19. The use of claim 18, wherein said pathogenic microorganism comprises yellow-white dysentery, asthma, erysipelas, oedema, clostridial enteritis, proliferative enteritis, tuberculosis, pasteurellosis, anthrax, salmonellosis.
20. use of a compound of formula (I) or a hydrate or pharmaceutically acceptable salt thereof as claimed in claim 1 or 2 in the manufacture of a medicament for reducing the level of IL-1 β and/or IL-1 β proteins, which are inflammatory factors that arise as a result of bacterial infection.
21. Use of a compound of formula (I) as claimed in claim 1 or 2, or a hydrate or pharmaceutically acceptable salt thereof, for the manufacture of a medicament for altering the polarity of the cell membrane of a microorganism.
22. The use of claim 21, wherein the pathogenic microorganism is a pathogenic gram positive bacterium, gram negative bacterium in livestock, marine animals.
23. The use according to claim 22, wherein the gram-positive bacterium is selected from the group consisting of staphylococcus, streptococcus, erysipelothrix, mycobacterium, bacillus anthracis; and/or the gram-negative bacteria are selected from escherichia coli, pasteurella, riemerella, salmonella, haemophilus, brucella.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2017110710068 | 2017-11-03 | ||
| CN201711071006 | 2017-11-03 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108992437A true CN108992437A (en) | 2018-12-14 |
| CN108992437B CN108992437B (en) | 2021-10-12 |
Family
ID=64537992
Family Applications (3)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810648982.3A Active CN108976151B (en) | 2017-11-03 | 2018-06-22 | Lauroyl arginine ethyl ester derivatives and use as antibacterial agents for animals |
| CN202111142030.2A Pending CN113797189A (en) | 2017-11-03 | 2018-06-22 | Lauroyl arginine ethyl ester derivatives and use as antibacterial agents for animals |
| CN201810649050.0A Active CN108992437B (en) | 2017-11-03 | 2018-06-22 | Use of lauroyl arginine ethyl ester as veterinary antibacterial agent |
Family Applications Before (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810648982.3A Active CN108976151B (en) | 2017-11-03 | 2018-06-22 | Lauroyl arginine ethyl ester derivatives and use as antibacterial agents for animals |
| CN202111142030.2A Pending CN113797189A (en) | 2017-11-03 | 2018-06-22 | Lauroyl arginine ethyl ester derivatives and use as antibacterial agents for animals |
Country Status (1)
| Country | Link |
|---|---|
| CN (3) | CN108976151B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114617218A (en) * | 2022-03-31 | 2022-06-14 | 南京财经大学 | Cyclodextrin-based lauroyl arginine ethyl ester inclusion compound and preparation method and application thereof |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030049305A1 (en) * | 2001-08-09 | 2003-03-13 | Gert-Wolfhard Von Rymon Lipinski | Preserved feedstuffs and process for their production |
| CN101227884A (en) * | 2005-08-01 | 2008-07-23 | 米雷特实验室股份公司 | Corrosion protection system including cationic surfactant |
| CN101500552A (en) * | 2006-08-03 | 2009-08-05 | 米雷特实验室股份公司 | Antiviral use of cationic surfactant |
| CN102088858A (en) * | 2008-07-02 | 2011-06-08 | 米雷特实验室股份公司 | Use of cationic surfactants as sporicidal agents |
| CN105212013A (en) * | 2015-08-27 | 2016-01-06 | 武汉志邦化学技术有限公司 | A kind of new Type of Preservatives and preparation technology thereof |
| CN106588701A (en) * | 2016-12-02 | 2017-04-26 | 南京华狮新材料有限公司 | Amino acid derivative and preparation method and application thereof |
| CN106665636A (en) * | 2015-11-07 | 2017-05-17 | 郑州明德生物科技有限公司 | Method for inhibiting contaminative microorganism during alcoholic fermentation by adopting ethyl lauroyl arginate |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090318557A1 (en) * | 2003-12-22 | 2009-12-24 | Stockel Richard F | Dermatological compositions |
| US9023891B2 (en) * | 2008-05-29 | 2015-05-05 | Nevada Naturals, Inc. | Synergistic antimicrobial agents |
| CN106565546A (en) * | 2016-10-21 | 2017-04-19 | 武汉桀升生物科技有限公司 | Lauroyl arginine ethyl ester glycol acid salt and preparation method and application thereof |
-
2018
- 2018-06-22 CN CN201810648982.3A patent/CN108976151B/en active Active
- 2018-06-22 CN CN202111142030.2A patent/CN113797189A/en active Pending
- 2018-06-22 CN CN201810649050.0A patent/CN108992437B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030049305A1 (en) * | 2001-08-09 | 2003-03-13 | Gert-Wolfhard Von Rymon Lipinski | Preserved feedstuffs and process for their production |
| CN101227884A (en) * | 2005-08-01 | 2008-07-23 | 米雷特实验室股份公司 | Corrosion protection system including cationic surfactant |
| CN101500552A (en) * | 2006-08-03 | 2009-08-05 | 米雷特实验室股份公司 | Antiviral use of cationic surfactant |
| CN102088858A (en) * | 2008-07-02 | 2011-06-08 | 米雷特实验室股份公司 | Use of cationic surfactants as sporicidal agents |
| CN105212013A (en) * | 2015-08-27 | 2016-01-06 | 武汉志邦化学技术有限公司 | A kind of new Type of Preservatives and preparation technology thereof |
| CN106665636A (en) * | 2015-11-07 | 2017-05-17 | 郑州明德生物科技有限公司 | Method for inhibiting contaminative microorganism during alcoholic fermentation by adopting ethyl lauroyl arginate |
| CN106588701A (en) * | 2016-12-02 | 2017-04-26 | 南京华狮新材料有限公司 | Amino acid derivative and preparation method and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| 沈强等主编: "《实用猪病防治技术》", 30 June 2012, 上海科学技术出版社 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108976151A (en) | 2018-12-11 |
| CN113797189A (en) | 2021-12-17 |
| CN108992437B (en) | 2021-10-12 |
| CN108976151B (en) | 2021-12-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| RU2484818C2 (en) | Lactylates for preventing and treating infections caused by gram-positive bacteria in animals | |
| FI114852B (en) | Acidified nitrate for use as an antimicrobial agent | |
| US20130018106A1 (en) | Hop acids as a replacement for antibiotics in animal feed | |
| KR101309583B1 (en) | Composition for preventing chicken contagious disease | |
| KR100707988B1 (en) | Complex antibiotic composition for bovine mastitis | |
| CN108992437B (en) | Use of lauroyl arginine ethyl ester as veterinary antibacterial agent | |
| CN108740356B (en) | Use of lauroyl arginine ethyl ester derivative as feed additive | |
| RU2197237C2 (en) | Method for treating animal diseases induced by bacteria (variants) and method for manufacturing a medicinal product | |
| US20160030475A1 (en) | Use of a clay product or a clay blend product to decrease the effects of bacterial disease in shrimp | |
| JPH0899884A (en) | Suppressant for proliferation of gram-negative bacterium | |
| CN108935967A (en) | Purposes of the lauroyl arginine ethyl ester as feed nutrition energy matter | |
| KR100438209B1 (en) | Complex antimicrobial composition based on carvacrol, thymol, and citral | |
| US7476379B1 (en) | Emu-based formulations for the treatment of damaged skin by inhibiting microbial and fungal activity | |
| TW200423950A (en) | Antidiarrheal composition | |
| JP2813471B2 (en) | Crude drug-containing feed | |
| Ellakany et al. | Studies on the effects of enrofloxacin overdose on different health parameters in broiler chickens | |
| CN109620877A (en) | Pure Chinese medicine fowl antibacterium disease preparation and preparation method thereof and application method | |
| KR20060027799A (en) | Aivlosin for the treatment of disease due to brachyspira pilosicoli or ornithobacterium rhinotracheale | |
| KR102208837B1 (en) | Composition for inhibiting adhesion, invasion of bacteria or antibacterial resistance comprising methyl gallate and fluoroquinolone antibacterial agent | |
| RU2356543C1 (en) | Preparation "gental" for treatment of gastrointestinal, respiratory diseases in calves and piglets of bacterial etiology and method of treating gastrointestinal, respiratory diseases in calves and piglets of bacterial etiology | |
| KR100307187B1 (en) | Livestock disease prevention and treatment using alginate oligosaccharide derivative | |
| RU2514647C1 (en) | Composition for treatment of infections of bacterial etiology in animals | |
| RU2166324C2 (en) | Veterinary probiotic preparation | |
| RU1826904C (en) | Method for prophylaxis and treatment of bartonellosis at calves | |
| CN109985022A (en) | Acu-Dyne and its preparation method and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |