JPS6159611B2 - - Google Patents
Info
- Publication number
- JPS6159611B2 JPS6159611B2 JP56108015A JP10801581A JPS6159611B2 JP S6159611 B2 JPS6159611 B2 JP S6159611B2 JP 56108015 A JP56108015 A JP 56108015A JP 10801581 A JP10801581 A JP 10801581A JP S6159611 B2 JPS6159611 B2 JP S6159611B2
- Authority
- JP
- Japan
- Prior art keywords
- bacteria
- test
- positive
- medium
- gallstones
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired
Links
- 241000894006 Bacteria Species 0.000 claims description 53
- 201000001883 cholelithiasis Diseases 0.000 claims description 22
- 210000003608 fece Anatomy 0.000 claims description 8
- 239000003814 drug Substances 0.000 claims description 6
- 210000000936 intestine Anatomy 0.000 claims description 6
- 229940079593 drug Drugs 0.000 claims description 4
- 241001465754 Metazoa Species 0.000 claims description 3
- 239000002075 main ingredient Substances 0.000 claims description 3
- 208000001130 gallstones Diseases 0.000 description 31
- 238000012360 testing method Methods 0.000 description 29
- 239000002609 medium Substances 0.000 description 26
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 16
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 16
- 241000699670 Mus sp. Species 0.000 description 12
- 230000001580 bacterial effect Effects 0.000 description 12
- 210000004027 cell Anatomy 0.000 description 12
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- 230000015572 biosynthetic process Effects 0.000 description 9
- 235000012000 cholesterol Nutrition 0.000 description 8
- 239000004310 lactic acid Substances 0.000 description 8
- 235000014655 lactic acid Nutrition 0.000 description 8
- 229920002472 Starch Polymers 0.000 description 7
- 238000004519 manufacturing process Methods 0.000 description 7
- 238000002360 preparation method Methods 0.000 description 7
- 239000008213 purified water Substances 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 239000008107 starch Substances 0.000 description 7
- 235000019698 starch Nutrition 0.000 description 7
- 238000003794 Gram staining Methods 0.000 description 6
- JVTAAEKCZFNVCJ-REOHCLBHSA-N L-lactic acid Chemical compound C[C@H](O)C(O)=O JVTAAEKCZFNVCJ-REOHCLBHSA-N 0.000 description 6
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 6
- 241000699666 Mus <mouse, genus> Species 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 238000012258 culturing Methods 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 239000008101 lactose Substances 0.000 description 6
- 229920001817 Agar Polymers 0.000 description 5
- 206010012735 Diarrhoea Diseases 0.000 description 5
- 241000194032 Enterococcus faecalis Species 0.000 description 5
- 241000186660 Lactobacillus Species 0.000 description 5
- 239000008272 agar Substances 0.000 description 5
- 229940039696 lactobacillus Drugs 0.000 description 5
- 241000894007 species Species 0.000 description 5
- 241000606124 Bacteroides fragilis Species 0.000 description 4
- 241000186000 Bifidobacterium Species 0.000 description 4
- 241000193403 Clostridium Species 0.000 description 4
- 241000193171 Clostridium butyricum Species 0.000 description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- SIKJAQJRHWYJAI-UHFFFAOYSA-N Indole Chemical compound C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 description 4
- 240000006024 Lactobacillus plantarum Species 0.000 description 4
- 235000013965 Lactobacillus plantarum Nutrition 0.000 description 4
- 229960000583 acetic acid Drugs 0.000 description 4
- 239000002775 capsule Substances 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 235000005911 diet Nutrition 0.000 description 4
- 230000037213 diet Effects 0.000 description 4
- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000000855 fermentation Methods 0.000 description 4
- 230000004151 fermentation Effects 0.000 description 4
- 210000003495 flagella Anatomy 0.000 description 4
- 239000007789 gas Substances 0.000 description 4
- 239000008187 granular material Substances 0.000 description 4
- 230000000968 intestinal effect Effects 0.000 description 4
- 229940072205 lactobacillus plantarum Drugs 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 4
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 241000186018 Bifidobacterium adolescentis Species 0.000 description 3
- 241001608472 Bifidobacterium longum Species 0.000 description 3
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 3
- KDYFGRWQOYBRFD-UHFFFAOYSA-N Succinic acid Natural products OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 3
- 241001147717 [Clostridium] sphenoides Species 0.000 description 3
- 235000011054 acetic acid Nutrition 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 229940009291 bifidobacterium longum Drugs 0.000 description 3
- 239000001913 cellulose Substances 0.000 description 3
- 229920002678 cellulose Polymers 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000007796 conventional method Methods 0.000 description 3
- 235000014113 dietary fatty acids Nutrition 0.000 description 3
- 239000000194 fatty acid Substances 0.000 description 3
- 229930195729 fatty acid Natural products 0.000 description 3
- 150000004665 fatty acids Chemical class 0.000 description 3
- 238000002245 gallstone dissolution Methods 0.000 description 3
- 239000006402 liver broth Substances 0.000 description 3
- 239000000523 sample Substances 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- NRHMKIHPTBHXPF-TUJRSCDTSA-M sodium cholate Chemical compound [Na+].C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC([O-])=O)C)[C@@]2(C)[C@@H](O)C1 NRHMKIHPTBHXPF-TUJRSCDTSA-M 0.000 description 3
- 239000008096 xylene Substances 0.000 description 3
- ZCVAOQKBXKSDMS-OIISXLGYSA-N (+)-trans-(R)-allethrin Chemical compound CC1(C)[C@H](C=C(C)C)[C@H]1C(=O)O[C@H]1C(C)=C(CC=C)C(=O)C1 ZCVAOQKBXKSDMS-OIISXLGYSA-N 0.000 description 2
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 2
- 241000606125 Bacteroides Species 0.000 description 2
- BPYKTIZUTYGOLE-IFADSCNNSA-N Bilirubin Chemical compound N1C(=O)C(C)=C(C=C)\C1=C\C1=C(C)C(CCC(O)=O)=C(CC2=C(C(C)=C(\C=C/3C(=C(C=C)C(=O)N\3)C)N2)CCC(O)=O)N1 BPYKTIZUTYGOLE-IFADSCNNSA-N 0.000 description 2
- JVTAAEKCZFNVCJ-UWTATZPHSA-N D-lactic acid Chemical compound C[C@@H](O)C(O)=O JVTAAEKCZFNVCJ-UWTATZPHSA-N 0.000 description 2
- RWSOTUBLDIXVET-UHFFFAOYSA-N Dihydrogen sulfide Chemical compound S RWSOTUBLDIXVET-UHFFFAOYSA-N 0.000 description 2
- 108010010803 Gelatin Proteins 0.000 description 2
- 206010060891 General symptom Diseases 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 241000509622 Mitsuokella multacida Species 0.000 description 2
- IOVCWXUNBOPUCH-UHFFFAOYSA-M Nitrite anion Chemical compound [O-]N=O IOVCWXUNBOPUCH-UHFFFAOYSA-M 0.000 description 2
- 241000194017 Streptococcus Species 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 235000019253 formic acid Nutrition 0.000 description 2
- 210000000232 gallbladder Anatomy 0.000 description 2
- 239000008273 gelatin Substances 0.000 description 2
- 229920000159 gelatin Polymers 0.000 description 2
- 235000019322 gelatine Nutrition 0.000 description 2
- 235000011852 gelatine desserts Nutrition 0.000 description 2
- 230000002949 hemolytic effect Effects 0.000 description 2
- 229910000037 hydrogen sulfide Inorganic materials 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- PZOUSPYUWWUPPK-UHFFFAOYSA-N indole Natural products CC1=CC=CC2=C1C=CN2 PZOUSPYUWWUPPK-UHFFFAOYSA-N 0.000 description 2
- RKJUIXBNRJVNHR-UHFFFAOYSA-N indolenine Natural products C1=CC=C2CC=NC2=C1 RKJUIXBNRJVNHR-UHFFFAOYSA-N 0.000 description 2
- 235000019359 magnesium stearate Nutrition 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000004899 motility Effects 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 235000019260 propionic acid Nutrition 0.000 description 2
- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 description 2
- 230000019086 sulfide ion homeostasis Effects 0.000 description 2
- 239000000454 talc Substances 0.000 description 2
- 229910052623 talc Inorganic materials 0.000 description 2
- 231100000820 toxicity test Toxicity 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- NQPDZGIKBAWPEJ-UHFFFAOYSA-N valeric acid Chemical compound CCCCC(O)=O NQPDZGIKBAWPEJ-UHFFFAOYSA-N 0.000 description 2
- WQZGKKKJIJFFOK-SVZMEOIVSA-N (+)-Galactose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-SVZMEOIVSA-N 0.000 description 1
- RUDATBOHQWOJDD-UHFFFAOYSA-N (3beta,5beta,7alpha)-3,7-Dihydroxycholan-24-oic acid Natural products OC1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)CC2 RUDATBOHQWOJDD-UHFFFAOYSA-N 0.000 description 1
- XYHKNCXZYYTLRG-UHFFFAOYSA-N 1h-imidazole-2-carbaldehyde Chemical compound O=CC1=NC=CN1 XYHKNCXZYYTLRG-UHFFFAOYSA-N 0.000 description 1
- ORRBHGDXBDFSFM-UHFFFAOYSA-N 1h-indole;nitric acid Chemical compound O[N+]([O-])=O.C1=CC=C2NC=CC2=C1 ORRBHGDXBDFSFM-UHFFFAOYSA-N 0.000 description 1
- IQUPABOKLQSFBK-UHFFFAOYSA-N 2-nitrophenol Chemical compound OC1=CC=CC=C1[N+]([O-])=O IQUPABOKLQSFBK-UHFFFAOYSA-N 0.000 description 1
- GWYFCOCPABKNJV-UHFFFAOYSA-M 3-Methylbutanoic acid Natural products CC(C)CC([O-])=O GWYFCOCPABKNJV-UHFFFAOYSA-M 0.000 description 1
- 238000012935 Averaging Methods 0.000 description 1
- 102100026189 Beta-galactosidase Human genes 0.000 description 1
- 241000185999 Bifidobacterium longum subsp. longum Species 0.000 description 1
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 1
- 102100035882 Catalase Human genes 0.000 description 1
- 108010053835 Catalase Proteins 0.000 description 1
- RGHNJXZEOKUKBD-UHFFFAOYSA-N D-gluconic acid Natural products OCC(O)C(O)C(O)C(O)C(O)=O RGHNJXZEOKUKBD-UHFFFAOYSA-N 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 241000588921 Enterobacteriaceae Species 0.000 description 1
- RGHNJXZEOKUKBD-SQOUGZDYSA-N Gluconic acid Natural products OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O RGHNJXZEOKUKBD-SQOUGZDYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 206010018910 Haemolysis Diseases 0.000 description 1
- 240000001929 Lactobacillus brevis Species 0.000 description 1
- 235000013957 Lactobacillus brevis Nutrition 0.000 description 1
- 102000004882 Lipase Human genes 0.000 description 1
- 108090001060 Lipase Proteins 0.000 description 1
- 239000004367 Lipase Substances 0.000 description 1
- 206010028813 Nausea Diseases 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 102000015439 Phospholipases Human genes 0.000 description 1
- 108010064785 Phospholipases Proteins 0.000 description 1
- 208000003251 Pruritus Diseases 0.000 description 1
- 244000061458 Solanum melongena Species 0.000 description 1
- 235000002597 Solanum melongena Nutrition 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 206010000059 abdominal discomfort Diseases 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 239000011609 ammonium molybdate Substances 0.000 description 1
- 235000018660 ammonium molybdate Nutrition 0.000 description 1
- APUPEJJSWDHEBO-UHFFFAOYSA-P ammonium molybdate Chemical compound [NH4+].[NH4+].[O-][Mo]([O-])(=O)=O APUPEJJSWDHEBO-UHFFFAOYSA-P 0.000 description 1
- 229940010552 ammonium molybdate Drugs 0.000 description 1
- GLMQHZPGHAPYIO-UHFFFAOYSA-L azanium;2-hydroxypropane-1,2,3-tricarboxylate;iron(2+) Chemical compound [NH4+].[Fe+2].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O GLMQHZPGHAPYIO-UHFFFAOYSA-L 0.000 description 1
- 108010005774 beta-Galactosidase Proteins 0.000 description 1
- GWYFCOCPABKNJV-UHFFFAOYSA-N beta-methyl-butyric acid Natural products CC(C)CC(O)=O GWYFCOCPABKNJV-UHFFFAOYSA-N 0.000 description 1
- 210000000941 bile Anatomy 0.000 description 1
- 239000003613 bile acid Substances 0.000 description 1
- 239000006161 blood agar Substances 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 229960001091 chenodeoxycholic acid Drugs 0.000 description 1
- RUDATBOHQWOJDD-BSWAIDMHSA-N chenodeoxycholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)CC1 RUDATBOHQWOJDD-BSWAIDMHSA-N 0.000 description 1
- 238000011260 co-administration Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 230000006735 deficit Effects 0.000 description 1
- 238000007872 degassing Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 239000012470 diluted sample Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 235000013345 egg yolk Nutrition 0.000 description 1
- 210000002969 egg yolk Anatomy 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 239000000174 gluconic acid Substances 0.000 description 1
- 235000012208 gluconic acid Nutrition 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000004313 iron ammonium citrate Substances 0.000 description 1
- 235000000011 iron ammonium citrate Nutrition 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 230000007803 itching Effects 0.000 description 1
- 235000019421 lipase Nutrition 0.000 description 1
- 229940057995 liquid paraffin Drugs 0.000 description 1
- 230000003908 liver function Effects 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000008693 nausea Effects 0.000 description 1
- 231100000957 no side effect Toxicity 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- UVGZJJKEELPWRH-JSCKKFHOSA-M potassium (2S,3S,4S,5R)-2,3,4,5-tetrahydroxy-6-oxohexanoate Chemical compound [K+].O=C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C([O-])=O UVGZJJKEELPWRH-JSCKKFHOSA-M 0.000 description 1
- 229940088417 precipitated calcium carbonate Drugs 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000001384 succinic acid Substances 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 235000021195 test diet Nutrition 0.000 description 1
- 229940124597 therapeutic agent Drugs 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 229940005605 valeric acid Drugs 0.000 description 1
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 1
- 229910000368 zinc sulfate Inorganic materials 0.000 description 1
- 239000011686 zinc sulphate Substances 0.000 description 1
- 235000009529 zinc sulphate Nutrition 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Description
本発明は、細菌を主剤とする胆石治療薬に関す
る。
胆石はコレステロール系胆石とビリルビン系胆
石とに大別されるが、近年わが国においても欧米
諸国と同様のレベルまでコレステロール系胆石が
増加しており、全胆石症中の70〜80％を占めるに
到つた。
したがつて、このコレステロール系胆石を手術
によらず溶解しようとする試みが多く報告されて
いる。最近、ケノデオキシコール酸等の胆汁酸を
経口投与することによるコレステロール系胆石溶
解療法が開発されているが、下痢と肝機能異常そ
の他に悪心、腹部不快感、掻痒感等の副作用が報
告されている。
本発明者は、上述のような副作用がない胆石症
治療薬について、鋭意研究した結果、特定の細菌
が胆石の発生防止と、治療に著効を奏することを
見い出し、それにもとづいて、本発明をなすに至
つた。
一般に、マウスの胆石形成にはコレステロール
とコール酸ソーダーの同時投与が必要である。そ
こで、腸内常在菌が全く欠除している無菌マウス
と自然マウスとを用いて実験胆石症を作成した。
その結果無菌マウス群で胆石形成食経口摂取４週
後にすでに全例に胆石の形成が見られるのに対
し、自然マウス群では４週、８週後で60％、24週
後にいたつて全例に胆石の形成が見られた。
これらの知見から、腸内常在菌が胆石形成に関
与しているものと推定された。
すなわち、本発明は、動物又は人の糞便より分
離した腸内常在菌及び腸内発育可能菌を主剤とす
る胆石症治療薬を提供するもので、本発明によれ
ば、副作用を全く生ずることなしに胆石の発生と
治療を効果的に行なうことができる。
本発明で用いる、上記腸内常在菌と腸内発育可
能菌は、例えば、人の糞便より分離したクロスト
リデユム・ブチリクム（Clostridium
butyricum）、クロストリデユム・スフエノイデ
ス（Clostridium sphenoides）、ICR系自然マウ
ス糞便より分離したバクテロイデス・フラギリス
（Bacteroides fragilis）、バクテロイデス・マル
チアシダス（Bacteroides multiacidus）、ビフイ
ドバクテリウム・アドレツセンテイス
（Bifidobacterium adolescentis）、ビフイドバク
テリウム・ロングム（Bifidobacterium
longum）、ラクトバチルス・ブレビス
（Lactobacillus brevis）、ラクトバチルス・プラ
ンタラム（Lactobacillus plantarum）、イシリシ
ア・コリイ（Esherichi coli）、ストレプトコツカ
ス・フエカリス（Streptococcus faecalis）であ
る。
これらの菌は、いずれも公知のものであつて
（バアジイズ マニユアル オブ デタアミネテ
イブ バクテリオロジ（Bergeys Manual of
Detrminative Bacterilology）参照）、次の方法に
よつて糞便から分離し、培養した。
糞便（試料）１ｇに９mlの生理食塩液を加えよ
く懸濁する。このように懸濁した液を10-1稀釈液
として、順次10倍段階稀釈を行なう。試料を適当
に稀釈し、その0.05mlを下記の選択平板に塗布
し、それぞれの培養法により培養し菌を分離し
た。さらにその菌を分離培地で増殖させて供試菌
を得た。
TECHNICAL FIELD The present invention relates to a drug for treating gallstones containing bacteria as a main ingredient. Gallstones are broadly classified into cholesterol-based gallstones and bilirubin-based gallstones, and in recent years cholesterol-based gallstones have increased in Japan to levels similar to those in Western countries, accounting for 70-80% of all cholelithiasis cases. Ivy. Therefore, many attempts have been made to dissolve cholesterol-based gallstones without surgery. Recently, cholesterol-based gallstone dissolving therapy has been developed by orally administering bile acids such as chenodeoxycholic acid, but side effects such as diarrhea, abnormal liver function, and nausea, abdominal discomfort, and itching have been reported. As a result of intensive research into cholelithiasis treatment drugs that do not have the above-mentioned side effects, the present inventor discovered that certain bacteria are highly effective in preventing and treating gallstones.Based on this, the present inventor developed the present invention. I arrived at the eggplant. In general, co-administration of cholesterol and sodium cholate is required for gallstone formation in mice. Therefore, we created experimental cholelithiasis using germ-free mice and natural mice, which lack all indigenous bacteria in the intestine.
As a result, in the germ-free mouse group, gallstone formation was already observed in all mice after 4 weeks of oral intake of the gallstone-forming diet, whereas in the natural mouse group, gallstone formation occurred in 60% of the mice after 4 and 8 weeks, and in all mice after 24 weeks. Gallstone formation was observed. From these findings, it was assumed that resident intestinal bacteria are involved in gallstone formation. That is, the present invention provides a cholelithiasis treatment drug whose main ingredients are indigenous intestinal bacteria and bacteria that can grow in the intestine isolated from animal or human feces, and according to the present invention, it does not cause any side effects. The occurrence and treatment of gallstones can be effectively carried out without any treatment. The above-mentioned intestinal resident bacteria and intestinal viable bacteria used in the present invention are, for example, Clostridium butyricum isolated from human feces.
butyricum), Clostridium sphenoides, Bacteroides fragilis isolated from the feces of ICR natural mice, Bacteroides multiacidus, Bifidobacterium adolescentis, and Bifidobacterium adolescentis. Bifidobacterium
longum), Lactobacillus brevis, Lactobacillus plantarum, Esherichi coli, and Streptococcus faecalis. All of these bacteria are known (Bergeys Manual of Determinative Bacteriology).
Detrminative Bacterilology) was isolated from feces and cultured by the following method. Add 9 ml of physiological saline to 1 g of feces (sample) and suspend well. The thus-suspended solution is used as a 10 -1 dilution solution, and serial dilutions of 10 times are carried out. The sample was diluted appropriately, and 0.05 ml of the diluted sample was applied to the following selective plate, cultured using each culture method, and bacteria were isolated. Furthermore, the bacteria were grown in an isolation medium to obtain test bacteria.
【表】【table】
【表】
得られた菌を主剤として、慣用の方法によつて
治療薬を製造した。
本発明による実験成績の一例を次に示す。
Ａ 胆石溶解実験
自然マウスを胆石形成食（１％コレステロール
と0.5％コール酸ソーダーを市販の粉末普通飼料
に添加した）で23日間飼育し、胆石形成後各実験
群にそれぞれの供試飼料を与えて18日目、32日目
に胆石の溶解状態を判定した。
１ 供試動物
ICR系自然マウス
２ 供試菌株
上記菌株を用いた。
３ 供試菌の性状は次のとおりである。
属（Genus）
(1) クロストリデユム（Clostridium）
嫌気性菌で胞子形成能を有する。グラム染色陽
性又は陰性、細菌の形態は直桿（Straight
rods）、又はやゝわん曲した桿菌（Slightly
rods）。
(2) バクテイロイデイス（Bacteroides）
嫌気性菌で胞子形成能を有しない。グラム染色
陰性、細菌の形態は直桿菌（Stright rods）、鞭
毛を有しない。発育過程においてコハク酸、酢
酸、乳酸、プロピオン酸、蟻酸を産生する。
(3) ビフイドバクテリウム（Bifidobacterium）
嫌気性菌で胞子形成能を有しない。グラム染色
陽性、細菌の形態は多形性桿菌
（diphtheroids）、鞭毛を有しない。発育過程にお
いて酢酸、乳酸を産生する。
(4) ラクトバチルス（Lactobacillus）
通性嫌気菌で胞子形成能を有しない。グラム染
色陽性。細菌の形態は直桿菌（Straight rods）
又は多形性桿菌（Diphtheroids）。鞭毛を有しな
い。発育過程において乳酸を産生する。
(5) イシリシイ（Escherichi）
好気性菌で運動性があり胞子形成能を有しな
い。グラム染色陰性、細菌の形態は直桿菌
（Straight rods）、鞭毛を有し、発育過程におい
てインドールを産生し、乳糖を分解してガスを発
生する。
(6) ストレプトコツカス（Streptococcus）
通性嫌気性菌で胞子形成能を有しない。グラム
染色は陽性。細菌の形態球菌（Cocci）。発育過
程に乳酸を産生する。
種 （Species）
菌種としては、例えばバクテロイデイス・フラ
ギリス（Bacteroides fragilis）、バクテロイデイ
ス・マルテアシダス（Bacteroides
multiacidus）、イシリシイ・コリイ（Escherichi
coli）、ビフイドバクテリウム・アドレセンテイ
ス（Bifidobacterium adolescentis）、ビフイドバ
クテリウム・ロングム（Bifidobacterium
longum）、クロストリデユム・ブチルクム
（Clostridium butyricum）、クロストリデユム・
スフエノイデス（Clostridium sphenoides）、ラ
クトバチルス・ブレビイス（Lactobacillus
brevis）、ラクトバチルス・プランタラム
（Lactobacillus plantarum）、ストレプトコツカ
ス・フエカリス（Streptococcus faecalis）を用
いることができ、その他上記属に入る菌種はすべ
て用いることができる。
ａ 種（Speies）の同定は次の方法によつた。
(1) 胆汁培地での発育
PYFG培地にOxgall（Difco）を２％加えた
培地に被検菌を接種し、同時にPYFG培地に菌
を接種したものを対照として発育を比較し、発
育の促進、不変、抑制、阻止を観察する。
(2) 硫化水素産生
H2S培地に菌を穿刺培養後、黒変したものを
陽性とする。
(3) 運動性
硫化水素産性試験に用いたものと同じ試験管
について運動性の有無を観察する。
(4) 硝酸塩還元
Indole−Nitrate培地（BBL）に菌を接種し、
２〜３日間培養後、亜硝酸試薬A1mlと亜硝酸
試薬B0.5mlを加え、赤色となつたものを陽性と
する。２〜５分間のうちに発色しないものにつ
いては亜鉛末を少量添加し、赤色となつたもの
は陰性とし、発色しなかつたものは陽性（完全
還元）とする。
(5) インドール産生
(2)，(3)項に用いたH2S培地に穿刺培養したも
の２mlに対し１mlのキシレンを加えてよく振盪
し、２分間放置後Ehrlich試薬を0.5ml添加し、
15分間以内にキシレンの層が赤色〜桃色となつ
たものを陽性とし、無色〜黄色となつたものを
陰性とする。
(6) Gelatin液化
Gelatin培地に菌を接種し、14日間培養後、
冷蔵庫中に静置し、固まつたものを陽性、再び
室温に出した後、30分以内に液状となつたもの
を弱陽性とする。
(7) 脂肪酸の産生
PYFG培地に菌を接種し、十分増殖するまで
培養（通常２〜７日）、同じ培地の非接種培地
を対照としてガスクロマトグラフイーにより、
ギ酸、醋酸、プロピオン酸、酩酸、イソ酩酸、
吉草酸、イソ吉草酸、カブロン酸などの揮発性
脂肪酸と乳酸、コハク酸などの不揮発性脂肪酸
を測定する。
(8) 炭水化物の発酵
発酵試験培地に菌を接種し、７日間培養後、
培地のPHを測定し、糖を加えていない培地に菌
を接種したもののPHを対照として判定する。PH
6.0以上は陰性（−）、PH5.5〜5.9を弱陽性、PH
5.4以下を陽性とする。なお、多検体（18検
体）のPHを自動測定記録する機器を用いると便
利である。
LactobacillusとBifidobacteriumについて
は、LB発酵試験培地を用い、PHメーターによ
らず、培養後培地が黄変したものを陽性、やや
黄変したものを弱陽性、わずかに黄味を帯びた
ものと赤紫色のものは陰性と判定してもよい。
(9) デンプンの加水分解
(7)項のデンプン培地に菌を接種・培養後、PH
測定の終つたものに、ルゴール液の１滴を加
え、無色（淡黄色）のものを陽性、青紫色とな
つたものを陰性とする。はじめ着色し数秒後に
無色となるものも陰性とする。
(10) エクスリンの加水分解
(8)項の発酵試験培地のエクスリン培地に菌を
接種・培養後、PHを測定したものについて１％
クエン酸鉄アンモニウム水溶液を２〜３滴加
え、黒色になつたものを陽性とする。
(11) レシチナーゼ反応
卵黄寒天平板に菌を接種し、２〜３日間培養
後、集落の周囲に淡黄色白色の不透明帯を生じ
たものを陽性とする。
(12) カタラーゼ産生
(11)項と同一の平板培養菌について、培養完了
後、約30分間空気に暴露した後の集落上に３％
H2O2水溶液を滴下し、発泡するものを陽性と
する。
(13) ブドウ糖からのガス産生
通性嫌気性Lactobacillusおよび
Bifidobacteriumの場合は、ダルハム管のはい
つた小試験管にBriggs liver brothを分注し、
100℃でガス抜きし滅菌した後、菌を接種し、
流動パラフインを重層し、７日間培養する。ダ
ルハム管の中にガスがたまつたものを陽性と判
定する。
嫌気性球菌の場合はBacto−Agar（Difco）
を1.5％加えたPYFG培地を溶かし、50℃に冷
し、菌を接種・培養し、ガス産生によつて寒天
にき裂の入つたものを陽性とする。
(14) 15℃発育
Briggs liver brothに0.15％の寒天を加えた
培地に菌を接種し、15℃、７日間培養後発育の
有無を判定する。Lactobacillusの分類に用い
る。
(15) 乳酸の旋光性
OR培地に菌を接種し、十分増殖するまで培
養（通常２〜３日）、その１mlを100mlのメスフ
ラスコにとり、0.5N NaOH0.5mlを加えよく振
つてから約６mlの精製水を加え、10％ZnSO4・
7H2O溶液の0.5mlをゆつくりと加えてよく振つ
て混合する。精製水で総量を正確に10mlとし、
遠心管に移しかえて約15分間放置した後遠沈
（300rpm10分）し、この上清を液とする。
液１mlを共栓試験管にとり、精製水10mlを
加えて混合、同時に調整したばかりの１％
FeCl3・6H2O（FeCl3・6H2O1ｇ、精製水95
ml，1N HCl5mlを混合溶解したもの）を加えて
混合、同様に処理した水を対照としてO.
D.400mμで測定し、あらかじめ作成しておい
た標準曲線により乳酸の総量を求める。
NAD（Sigma Grade ）125mgにGlycine
buffer（Sigma−No.826−３，pH9.2）75mlを
加えた後、Ｌ（＋）−LDH（Sigma Type）
0.2mlおよび精製水4.8mlを加え混合する。これ
を３mlずつ２本の共栓試験管に分け、上記１液
４μずつをそれぞれの試験管に加えて混合、
37℃，60分間保持した後、O.D.340mμで比色
定量し、あらかじめ作成しておいた標準曲線か
らＬ（＋）乳酸の値を求める。
Ｄ（−）乳酸の測定は、NAD（Sigma
Grade）90mgにGlycine buffer 54mlを加え、
Ｄ（−）−LDH1mlおよび精製水2.5mlを加えた
後、Ｌ（＋）乳酸の場合と同様、Ｄ（−）乳酸
の値を求める。
以上の値から、総乳酸量に対するＬ（＋）乳
酸の％を求め、０〜20％→Ｄ（−），20〜40％
→Ｄ＋DL，40〜60％→DL，60〜80％→Ｌ＋
DL，80〜100％→Ｌ（＋）と判定する。
(16) リパーゼ反応
(11)項と同一の平板について、集落の表面およ
びその周囲のせまい範囲に真珠様の層のみられ
るものを陽性とする。あるいはTrybutyrin寒
天平板で白濁を生ずるものを陽性としてもよ
い。
(17) グルコン酸分解
KH2SO42ｇ，MgSO45H2O0.1ｇ，NaCl5ｇ，
（NH4）2SO40.5ｇ、グルクロン酸カリウム10
ｇ、精製水１を加えた培地（PH7.5）に菌を
接種し、37℃３日間嫌気性培養する。判定は中
試験管に培養液１mlを採り、１％モリブデン酸
アンモニウム３ml、氷酢酸0.2mlを加え湯浴中
で５分間煮沸し、冷却後、液が濃い青色ならば
陽性、薄い青色または無色ならば陰性と判定す
る。
(18) 溶血反応
血液寒天平板上に塗抹して培養した場合、溶
血帯を生ずるならば陽性、溶血帯を生じないな
らば陰性と判定する。
(19) オキシダーゼ産生
１％のトリメチル−Ｐ−フエニレンデイアミ
ン（Trimethyl−Ｐ−Phenylenediamine）溶液
を平板培地の集落の上にかけて、紫色になるも
のを陽性と判定する。
(20) β−ガラクトシダーゼ産生
ONPG−（Ｏ−Nitrophenyl−β−Ｄ−
Galactopyranoside）を含むペプトン水中で菌
を１夜培養する。無色の基質が黄色のＯ−ニト
ロフエノールに加水分解されるものを陽性とす
る。
(21) 60℃30分間耐熱性
ブリツクス・リバー・ブロス（Briggs liver
broth）に菌を接種後、試験管にゴム栓をし
て、全体を恒温槽にうずめ、60℃30分間加熱後
の菌の発育したものを陽性とする。
(22) ４％NaCl中の発育
ヴイヨン（Bouillon）培地に、NaClを４％加
え、菌を接種後、発育したものを陽性とする。
(23) PH9.6中の発育
ヴイヨン（Bouillon）培地を、10％NaOHで
PH9.6に調整し、菌を接種後、発育したものを
陽性とする。
ｂ 種（Species）の性状は次のとおりである。[Table] Using the obtained bacteria as a main agent, a therapeutic drug was produced by a conventional method. An example of experimental results according to the present invention is shown below. A. Gallstone dissolution experiment Natural mice were kept on a gallstone-forming diet (1% cholesterol and 0.5% sodium cholate added to commercially available powdered normal feed) for 23 days, and after gallstone formation, each experimental group was given the respective test diet. The dissolution state of the gallstones was determined on the 18th and 32nd days. 1 Test animal ICR natural mouse 2 Test strain The above strains were used. 3 The properties of the test bacteria are as follows. Genus (1) Clostridium An anaerobic bacterium with spore-forming ability. Gram staining is positive or negative, and the morphology of the bacteria is straight rod.
rods), or slightly bent rods (slightly
rods). (2) Bacteroides An anaerobic bacterium that does not have spore-forming ability. Gram staining is negative, the morphology of the bacteria is straight rods, and there are no flagella. During development, it produces succinic acid, acetic acid, lactic acid, propionic acid, and formic acid. (3) Bifidobacterium An anaerobic bacterium that does not have spore-forming ability. Gram staining is positive, the bacterial morphology is pleomorphic rods (diphtheroids), and there are no flagella. During the development process, it produces acetic acid and lactic acid. (4) Lactobacillus A facultative anaerobe that does not have spore-forming ability. Positive Gram staining. The morphology of the bacteria is Straight rods.
or Diphtheroids. It has no flagella. Produces lactic acid during development. (5) Escherichi Escherichi is an aerobic bacterium that is motile and has no spore-forming ability. Gram staining is negative, the bacterial morphology is straight rods, it has flagella, produces indole during the growth process, and decomposes lactose to generate gas. (6) Streptococcus A facultative anaerobe that does not have spore-forming ability. Gram staining was positive. Bacterial form Cocci (Cocci). Produces lactic acid during development. Species Examples of bacterial species include Bacteroides fragilis, Bacteroides malteacidus, and Bacteroides fragilis.
multiacidus), Escherichi coryi
coli), Bifidobacterium adolescentis, Bifidobacterium longum
longum), Clostridium butyricum, Clostridium butyricum
Clostridium sphenoides, Lactobacillus brebis
brevis), Lactobacillus plantarum, and Streptococcus faecalis, and all other bacterial species that belong to the above genera can be used. a Species (Speies) were identified by the following method. (1) Growth in bile medium The test bacteria were inoculated into a PYFG medium with 2% Oxgall (Difco) added, and the growth was compared with a control in which the bacteria were inoculated into a PYFG medium. Observe constancy, suppression, inhibition. (2) Hydrogen sulfide production After pricking and culturing the bacteria in H 2 S medium, those that turn black are considered positive. (3) Motility Observe the presence or absence of motility in the same test tube used for the hydrogen sulfide production test. (4) Inoculate the bacteria into nitrate-reducing Indole-Nitrate medium (BBL),
After culturing for 2 to 3 days, add 1 ml of nitrite reagent A and 0.5 ml of nitrite reagent B, and if the color turns red, it is considered positive. For those that do not develop color within 2 to 5 minutes, add a small amount of zinc powder, and those that turn red are considered negative, and those that do not develop color are considered positive (complete reduction). (5) Indole production Add 1 ml of xylene to 2 ml of the puncture culture in the H 2 S medium used in sections (2) and (3), shake well, leave for 2 minutes, then add 0.5 ml of Ehrlich reagent.
The test is considered positive if the xylene layer turns red to pink within 15 minutes, and negative if the xylene layer turns colorless to yellow within 15 minutes. (6) Gelatin liquefaction Inoculate bacteria into Gelatin medium and culture for 14 days.
If the test material solidifies after being left in the refrigerator, it is considered positive; if it becomes liquid within 30 minutes after returning to room temperature, it is considered weakly positive. (7) Production of fatty acids Inoculate bacteria into PYFG medium, culture until sufficient growth (usually 2 to 7 days), and perform gas chromatography using the same medium without inoculation as a control.
Formic acid, acetic acid, propionic acid, oxic acid, isocarboxylic acid,
Measure volatile fatty acids such as valeric acid, isovaleric acid, and cabroic acid, and non-volatile fatty acids such as lactic acid and succinic acid. (8) Fermentation of carbohydrates Bacteria were inoculated into fermentation test medium, and after culturing for 7 days,
Measure the PH of the medium and use the PH of a medium inoculated with bacteria to which no sugar has been added as a control. PH
6.0 or higher is negative (-), PH5.5-5.9 is weakly positive, PH
5.4 or less is considered positive. Note that it is convenient to use a device that automatically measures and records the PH of multiple samples (18 samples). For Lactobacillus and Bifidobacterium, use LB fermentation test medium, regardless of the PH meter, positive if the medium turns yellow after culture, weak positive if slightly yellow, and red-purple if slightly yellowish. If the result is positive, it may be considered negative. (9) Hydrolysis of starch After inoculating and culturing the bacteria in the starch medium described in (7), the pH
One drop of Lugol's solution is added to the sample after the measurement is completed, and if it is colorless (pale yellow), it is considered positive, and if it becomes bluish-purple, it is considered negative. Tests that are initially colored but become colorless after a few seconds are also considered negative. (10) Hydrolysis of Exrin After inoculating and culturing the bacteria in the Exrin medium of the fermentation test medium in Section (8), 1% of the PH was measured.
Add 2 to 3 drops of iron ammonium citrate aqueous solution, and if it turns black, it is considered positive. (11) Lecithinase reaction After inoculating the bacteria onto an egg yolk agar plate and incubating for 2 to 3 days, a positive test is taken if a pale yellowish white opaque zone is formed around the colony. (12) Catalase production For the same plate-cultured bacteria as in section (11), 3% of
Add H 2 O 2 aqueous solution dropwise, and if it bubbles, it is considered positive. (13) Gas production from glucose Facultatively anaerobic Lactobacillus and
For Bifidobacterium, dispense Briggs liver broth into a small test tube with a Dalham tube.
After degassing and sterilizing at 100℃, inoculate with bacteria,
Overlay liquid paraffin and culture for 7 days. A test result is determined to be positive if gas has accumulated in the Durham tube. Bacto−Agar (Difco) for anaerobic cocci
Dissolve PYFG medium to which 1.5% is added, cool to 50℃, inoculate and culture the bacteria, and test as positive if the agar cracks due to gas production. (14) Growth at 15°C Bacteria are inoculated into a medium containing Briggs liver broth with 0.15% agar added, and after culturing at 15°C for 7 days, the presence or absence of growth is determined. Used for classification of Lactobacillus. (15) Optical rotation of lactic acid Inoculate the bacteria in OR medium and culture until sufficient growth (usually 2 to 3 days). Transfer 1 ml of the bacteria to a 100 ml volumetric flask, add 0.5 ml of 0.5N NaOH, shake well, and approximately 6 ml. Add purified water and 10% ZnSO4 .
Gently add 0.5 ml of 7H 2 O solution and shake well to mix. Make the total volume exactly 10ml with purified water,
Transfer to a centrifuge tube, let stand for about 15 minutes, then centrifuge (300 rpm, 10 minutes), and use the supernatant as a liquid. Pour 1 ml of the solution into a stoppered test tube, add 10 ml of purified water, mix, and at the same time add the freshly prepared 1%
FeCl 3・6H 2 O (FeCl 3・6H 2 O1g, purified water 95
ml, mixed and dissolved with 5 ml of 1N HCl) and mixed, and similarly treated water was used as a control.
D. Measure at 400 mμ and determine the total amount of lactic acid using a standard curve prepared in advance. NAD (Sigma Grade) 125mg and Glycine
After adding 75ml of buffer (Sigma-No.826-3, pH9.2), L(+)-LDH (Sigma Type)
Add 0.2ml and 4.8ml of purified water and mix. Divide this into two stoppered test tubes of 3 ml each, add 4 μ of each of the above liquids to each test tube, and mix.
After holding at 37°C for 60 minutes, colorimetric determination is performed using OD340mμ, and the value of L(+) lactic acid is determined from a standard curve prepared in advance. Measurement of D(-)lactate is performed using NAD (Sigma
Add Glycine buffer 54ml to 90mg (Grade),
After adding 1 ml of D(-)-LDH and 2.5 ml of purified water, determine the value of D(-) lactic acid in the same way as for L(+) lactic acid. From the above values, calculate the percentage of L (+) lactic acid to the total amount of lactic acid, 0 to 20% → D (-), 20 to 40%
→D+DL, 40~60% →DL, 60~80% →L+
DL, 80-100% → Determined as L(+). (16) Lipase reaction Regarding the same plate as in item (11), if a pearl-like layer is observed on the surface of the colony and in a narrow area around it, the test is considered positive. Alternatively, those that produce white turbidity on a Trybutyrin agar plate may be considered positive. (17) Gluconic acid decomposition KH 2 SO 4 2g, MgSO 4 5H 2 O 0.1g, NaCl 5g,
(NH 4 ) 2 SO 4 0.5g, potassium glucuronate 10
g. Inoculate the bacteria into a medium (PH7.5) to which 1 part of purified water has been added, and culture anaerobically at 37°C for 3 days. Judgment is made by taking 1 ml of the culture solution in a medium test tube, adding 3 ml of 1% ammonium molybdate and 0.2 ml of glacial acetic acid, boiling it in a hot water bath for 5 minutes, and after cooling, if the liquid is dark blue, it is positive; if it is light blue or colorless, it is positive. If the test result is negative, the test result will be determined as negative. (18) Hemolytic reaction When smeared on a blood agar plate and cultured, if a hemolytic zone occurs, it is determined to be positive, and if no hemolytic zone occurs, it is determined to be negative. (19) Oxidase production A 1% Trimethyl-P-phenylenediamine solution is poured over the colonies on the plate medium, and those that turn purple are determined to be positive. (20) β-galactosidase production ONPG-(O-Nitrophenyl-β-D-
The bacteria are cultured overnight in peptone water containing (Galactopyranoside). A positive result is when a colorless substrate is hydrolyzed to yellow O-nitrophenol. (21) Heat resistant at 60°C for 30 minutes Briggs liver broth
After inoculating the bacteria into the test tube (broth), put a rubber stopper on the test tube, immerse the entire tube in a constant temperature bath, and test as positive if bacteria have grown after heating at 60°C for 30 minutes. (22) Growth in 4% NaCl 4% NaCl is added to Bouillon medium, and after inoculating the bacteria, those that grow are considered positive. (23) Growth in PH9.6 Bouillon medium was diluted with 10% NaOH.
After adjusting the pH to 9.6 and inoculating the bacteria, those that grow are considered positive. The properties of Species b are as follows.
【表】【table】
【表】【table】
【表】【table】
【表】
る。
[Table]
【表】【table】
【表】【table】
【表】【table】
【表】【table】
【表】【table】
【表】【table】
【表】【table】
【表】【table】
【表】
５ 胆石溶解の判定
下記の４段階により判定した。
−：胆石は全く見られず、その他の変化も見ら
れない。
＋：胆石は見られるが軽度である。
２＋：胆のうの1/3〜1/2に胆石がつまつている。
３＋：胆のうの1/2以上に胆石がつまつている。
この判定には少なくとも３人以上の個人の判定
を平均化して示した。
上記の判定の平均値をIndex（＋数の総和をマ
ウスの匹敵で割つた）として求めた。
その結果を表−１に示す。[Table] 5 Judgment of gallstone dissolution Judgment was made according to the following four stages. -: No gallstones or other changes observed. +: Gallstones are seen, but they are mild. 2+: 1/3 to 1/2 of the gallbladder is filled with gallstones. 3+: More than 1/2 of the gallbladder is filled with gallstones. This judgment was made by averaging the judgments of at least three individuals. The average value of the above judgments was determined as an Index (sum of + numbers divided by mouse comparison). The results are shown in Table-1.
【表】【table】
【表】
（一般症状の観察）
実験期間中、供試菌投与による軟便、下痢等の
副作用は認められなかつた。
Ｂ 胆石形成予防実験
ICR系無菌マウスに同系マウス糞便由来のイシ
リシイ・コリイ（Escherichi coli）、ストレプト
コツカス・フエカリス（Streptococcus
faecalis）、人の糞便由来のクロストリデユム・ブ
チリクム（Clostridium butyricum）を106〜108
オーダ／mlの懸濁液0.1mlを単独及び同時混合投
与し、定着させたのち、１％コレステロールと
0.5％コール酸ソーダーを市販の普通飼料に添加
した胆石形成食を高圧蒸気滅菌後自由に経口摂取
させた。
胆石形成食投与後、４週後胆石形成率（胆石形
成マウス数割る全マウス数掛ける100）をみた。
その結果を表−２に示す。[Table] (Observation of general symptoms) During the experiment period, no side effects such as loose stools or diarrhea were observed due to administration of the test bacteria. B Gallstone formation prevention experiment Escherichi coli (Escherichi coli) derived from syngeneic mouse feces and Streptococcus fuecalis (Streptococcus
faecalis), and Clostridium butyricum ( Clostridium butyricum) derived from human feces .
Administer 0.1 ml of the order/ml suspension alone or simultaneously, and after fixing, add 1% cholesterol.
A gallstone-forming diet in which 0.5% sodium cholic acid was added to a commercially available normal feed was sterilized by high-pressure steam and allowed to be orally ingested ad libitum. Four weeks after administration of the gallstone-forming diet, the gallstone formation rate (number of gallstone-forming mice divided by total number of mice multiplied by 100) was determined.
The results are shown in Table-2.
【表】
（一般症状の観察）
実験期間中、供試菌投与による軟便、下痢等は
認められなかつた。
上記の如く本発明により腸内常在菌及び腸内発
育可能菌を投与することにより副作用を防止して
胆石を溶解することが可能である。
本発明で用いる腸内菌は慣用の方法で乾燥菌体
を得たのち、錠剤、カプセル剤、丸剤、顆粒剤、
細粒剤のような各種の剤形に調整して人に投与す
ることができるが、その調整には乳糖、澱粉、セ
ルロース類、タルク等の医薬品に用いる賦形剤を
使用することができる。
本発明の腸内菌は１×108〜９×1010個／日の
用量で人に経口投与することができる。好ましく
は７×109〜２×1010個／日を投与するのが有効
である。
以下の調剤例は本発明の胆石症の治療薬を慣用
の方法で調整したものである。
調剤例１ （錠剤）
ビフイドバクテリウム・ロングムの 30mg
乾燥菌体 （３×109個含有）
乾燥澱粉 180mg
乳 糖 72mg
結晶セルロース 15mg
ステアリン酸マグネシウム 1.5mg
タルク 1.5mg
調剤例２ カプセル剤
クロストリジウムブチリカムの 80mg
乾燥菌体 （３×109個含有）
乾燥澱粉 20mg
乳 糖 50mg
結晶セルロース １mg
ステアリン酸マグネシウム 0.5mg
調剤例３ 細粒剤
ストレプトコツカスフエカリスの 40mg
乾燥菌体 （４×109個含有）
乳 糖 360mg
乾燥澱粉 50mg
沈降炭酸カルシウム 550mg
調剤例４ 顆粒剤
ラクトバシラス・プランタラムの 30mg
乾燥菌体 （３×109個含有）
乾燥澱粉 40mg
乳 糖 15mg
白 糖 15mg
毒性試験
前記乾燥菌末をマウスに経口投与してその毒性
を調べた。
１mg当り菌体が１×109個になるように調整し
た各種腸内菌の乾燥菌末を、マウスに技術的に投
与可能な最大量である20ｇ／ｇ強制経口投与した
ところ、クロストリジウム・ブチリカム、クロス
トリジウム・スフエノイデス、バクテロイデス・
フラギリス、バクテロイデス・マルチアシダス、
ビフイドバクテリウム・アドレセンテイス、ビフ
イドバクテリウム・ロングム、ラクトバシラス・
プランタラム、イシリシイ・コリイ及びストレプ
トコツカス・フエカリスについていずれもマウス
生存率100％であり、LD50（半致死量）は求めら
れず、極めて毒性の低いものであることがわかつ
た。上記胆石溶解実験、胆石形成予防実験および
毒性試験の結果は、本発明の腸内菌による臨床へ
の応用を可能にするものである。
以下にその臨床例をあげる。
調剤例２によるカプセル剤を用いた。即ち、コ
レステロール系胆石と思われるもので胆機能に著
るしい障害がなく、胆石の直径が10mm以下の患者
５例に１回３カプセル、１日３回、12カ月間報用
させ、Ｘ線造影所見により診断したところ２例が
消失し、１例が縮少した。消失期間は７〜８カ月
であつた。[Table] (Observation of general symptoms) During the experiment period, no loose stools, diarrhea, etc. were observed due to administration of the test bacteria. As described above, by administering indigenous bacteria in the intestine and bacteria capable of growing in the intestine according to the present invention, it is possible to prevent side effects and dissolve gallstones. The enteric bacteria used in the present invention is prepared by obtaining dry bacterial cells by a conventional method, and then preparing tablets, capsules, pills, granules, etc.
It can be prepared into various dosage forms such as fine granules and administered to humans, and excipients used in pharmaceuticals such as lactose, starch, cellulose, and talc can be used for the preparation. The enteric bacteria of the present invention can be orally administered to humans at a dose of 1 x 108 to 9 x 1010 cells/day. Preferably, it is effective to administer 7×10 9 to 2×10 10 cells/day. The following formulation examples are preparations of the therapeutic agent for cholelithiasis of the present invention using conventional methods. Preparation example 1 (tablet) 30 mg dried bacterial cells of Bifidobacterium longum (containing 3 x 10 9 cells) Dry starch 180 mg Lactose 72 mg Crystalline cellulose 15 mg Magnesium stearate 1.5 mg Talc 1.5 mg Preparation example 2 Capsule Clostridium butylicum 80mg dried bacterial cells (contains 3×10 9 cells) Dry starch 20mg Lactose 50mg Crystalline cellulose 1mg Magnesium stearate 0.5mg Preparation example 3 Fine granules 40mg dried bacterial cells of Streptococcus faecalis (contains 4×10 9 cells) ) Lactose 360 mg Dry starch 50 mg Precipitated calcium carbonate 550 mg Preparation example 4 Granules 30 mg dried bacterial cells of Lactobacillus plantarum (containing 3 x 10 9 cells) Dry starch 40 mg Lactose 15 mg White sugar 15 mg Toxicity test The dried bacterial powder was applied to mice. The toxicity was investigated by oral administration. When dried bacterial powder of various enteric bacteria adjusted to have 1 x 10 9 cells per mg was forcibly administered to mice at 20 g/g, the maximum amount that can be administered technically, Clostridium butylicum was detected. , Clostridium sphenoides, Bacteroides
fragilis, Bacteroides multiacidus,
Bifidobacterium adrecenteis, Bifidobacterium longum, Lactobacillus
Plantarum, I. coli, and Streptococcus faecalis all had a 100% mouse survival rate, and no LD50 (half-lethal dose) was required, indicating that they have extremely low toxicity. The results of the above-mentioned gallstone dissolution experiment, gallstone formation prevention experiment, and toxicity test enable the clinical application of the enterobacteriaceae of the present invention. A clinical example is given below. Capsules according to Preparation Example 2 were used. In other words, 5 patients with what appeared to be cholesterol-based gallstones, who had no significant impairment in biliary function and whose gallstones were 10 mm or less in diameter, were given 3 capsules at a time, 3 times a day for 12 months, and X-rays were administered. Diagnosis based on contrast findings revealed that 2 cases disappeared and 1 case shrank. The disappearance period was 7-8 months.
Claims (1)
び腸内発育可能菌を主剤とする胆石症治療薬。1. A drug for treating cholelithiasis whose main ingredient is indigenous bacteria in the intestine and bacteria that can grow in the intestine isolated from animal or human feces.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP56108015A JPS5810520A (en) | 1981-07-10 | 1981-07-10 | Remedy for hepatolithiasis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP56108015A JPS5810520A (en) | 1981-07-10 | 1981-07-10 | Remedy for hepatolithiasis |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPS5810520A JPS5810520A (en) | 1983-01-21 |
| JPS6159611B2 true JPS6159611B2 (en) | 1986-12-17 |
Family
ID=14473819
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP56108015A Granted JPS5810520A (en) | 1981-07-10 | 1981-07-10 | Remedy for hepatolithiasis |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPS5810520A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998036050A1 (en) * | 1997-02-14 | 1998-08-20 | Nippoh Chemicals Co., Ltd. | Clostridium butyricum having preventive and therapeutic effects on hepatopathy, and liver supporting agents, foods and feeds each containing the culture medium thereof |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| SE463598B (en) * | 1984-11-08 | 1990-12-17 | Chemical Dynamics Sweden Ab | PREPARATION, PROVIDED TO INCREASE THE ADMINISTRATION OF BACTERIA TO THE GIANT GAS OF HUMAN AND ANIMALS, AND A MANUFACTURING PROCEDURE FOR THIS |
| IT1309427B1 (en) * | 1999-05-28 | 2002-01-23 | Mendes S U R L | DIETARY OR PHARMACEUTICAL COMPOSITION USEFUL FOR THE PREVENTION OR TREATMENT OF HYPEROXIDE AND ITS USE |
-
1981
- 1981-07-10 JP JP56108015A patent/JPS5810520A/en active Granted
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1998036050A1 (en) * | 1997-02-14 | 1998-08-20 | Nippoh Chemicals Co., Ltd. | Clostridium butyricum having preventive and therapeutic effects on hepatopathy, and liver supporting agents, foods and feeds each containing the culture medium thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| JPS5810520A (en) | 1983-01-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP4580542B2 (en) | Microorganism for treating obesity or diabetes and pharmaceutical composition containing the microorganism | |
| JP5137571B2 (en) | Methods and compositions for dietary management of autoimmune disorders | |
| JP4350170B2 (en) | Pharmaceutical preparation containing Lactobacillus casei rhamnosus | |
| EP2040724B1 (en) | Biotherapeutic compositions comprising probiotic escherichia coli and metronidazole and uses thereof | |
| KR100446482B1 (en) | Epithelial adhesive lactobacilli | |
| JP2014001245A (en) | Block of pathogen by bacillus coagulans | |
| JPS61280433A (en) | Acidophilus strain culture, isolation and therapy by same bacteria | |
| US20040071680A1 (en) | Microorganisms which are taken effects in preventing and/or treating obesity or diabetes mellitus | |
| AU5784696A (en) | Pharmaceutical composition comprising lactobacillus plantarum and arginine | |
| KR100794702B1 (en) | Microorganisms which are taken effects in preventing and/or treating Obesity or Diabetes Mellitus | |
| JP2018532380A (en) | Enterobacteriaceae butyric acid intestini and uses thereof | |
| KR100794701B1 (en) | Microorganisms for Obesity or Diabetes Mellitus | |
| MXPA97001816A (en) | Bacterials and pharmaceutical composition containing such strains and using the same to prevent and treat diseases associated with metabolism altered biliary deactives or caused by my | |
| WO2024051180A1 (en) | Use of phenyllactic acid in inhibiting helicobacter pylori infection | |
| JPS6159611B2 (en) | ||
| Reid et al. | Urogenital microflora and urinary tract infections | |
| Gibson et al. | Recovery of a probiotic organism from human faeces after oral dosing | |
| WO2023184631A1 (en) | Akkermansia muciniphila and use thereof in preparing anti-tumor drug | |
| WO2018107364A1 (en) | Collinsella shenzhenensis and applications thereof | |
| NL2030011B1 (en) | Prevention or treatment of hepatic steatosis | |
| WO2023240936A1 (en) | Lactobacillus reuteri for prolonging lifespan, resisting aging and reducing fat, and product thereof and use thereof | |
| Al-Aabideen | Effect of Probiotic on Motility Factors and Swarming Phenomenon of Proteus mirabilis | |
| CN117645955A (en) | Eubacterium rectum with function of promoting recovery of intestinal epithelial cells and application thereof | |
| van Nuenen et al. | The effect of anti-TNF-α antibody therapy in Crohn’s disease on composition and activity of faecal microbiota: a pilot study | |
| JPS58107179A (en) | Exzyme inhibiting agent |