JP2012126690A - Simple method for producing chafurosides - Google Patents
Simple method for producing chafurosides Download PDFInfo
- Publication number
- JP2012126690A JP2012126690A JP2010281506A JP2010281506A JP2012126690A JP 2012126690 A JP2012126690 A JP 2012126690A JP 2010281506 A JP2010281506 A JP 2010281506A JP 2010281506 A JP2010281506 A JP 2010281506A JP 2012126690 A JP2012126690 A JP 2012126690A
- Authority
- JP
- Japan
- Prior art keywords
- chafuroside
- precursor
- chaflosides
- chafurosides
- reaction
- 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
- 229930185379 chafuroside Natural products 0.000 title claims abstract description 60
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 47
- 239000002243 precursor Substances 0.000 claims abstract description 77
- 238000010438 heat treatment Methods 0.000 claims abstract description 71
- 239000000203 mixture Substances 0.000 claims abstract description 33
- 239000002253 acid Substances 0.000 claims abstract description 27
- 238000000034 method Methods 0.000 claims abstract description 27
- 150000003839 salts Chemical class 0.000 claims abstract description 27
- 230000008569 process Effects 0.000 claims abstract description 6
- 239000007864 aqueous solution Substances 0.000 claims description 50
- DZQBNDGXEFWXEK-UHFFFAOYSA-N chafuroside A Natural products OC1C(O)C(CO)OC(C2=C(O)C=3C(=O)C=4)C1OC2=CC=3OC=4C1=CC=C(O)C=C1 DZQBNDGXEFWXEK-UHFFFAOYSA-N 0.000 claims description 39
- DZQBNDGXEFWXEK-ODJUOBDQSA-N chembl108032 Chemical compound O([C@@H]1[C@H](C2=C(O)C=3C(=O)C=4)O[C@@H]([C@H]([C@@H]1O)O)CO)C2=CC=3OC=4C1=CC=C(O)C=C1 DZQBNDGXEFWXEK-ODJUOBDQSA-N 0.000 claims description 39
- JMFSHKGXVSAJFY-UHFFFAOYSA-N Saponaretin Natural products OCC(O)C1OC(Oc2c(O)cc(O)c3C(=O)C=C(Oc23)c4ccc(O)cc4)C(O)C1O JMFSHKGXVSAJFY-UHFFFAOYSA-N 0.000 claims description 35
- MOZJVOCOKZLBQB-UHFFFAOYSA-N Vitexin Natural products OCC1OC(Oc2c(O)c(O)cc3C(=O)C=C(Oc23)c4ccc(O)cc4)C(O)C(O)C1O MOZJVOCOKZLBQB-UHFFFAOYSA-N 0.000 claims description 35
- MYXNWGACZJSMBT-VJXVFPJBSA-N isovitexin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1C1=C(O)C=C(OC(=CC2=O)C=3C=CC(O)=CC=3)C2=C1O MYXNWGACZJSMBT-VJXVFPJBSA-N 0.000 claims description 19
- OYJCWTROZCNWAA-UHFFFAOYSA-N isovitexin Natural products OCC1OC(C(O)C(O)C1O)c2c(O)cc3CC(=CC(=O)c3c2O)c4ccc(O)cc4 OYJCWTROZCNWAA-UHFFFAOYSA-N 0.000 claims description 19
- SGEWCQFRYRRZDC-VPRICQMDSA-N vitexin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1C1=C(O)C=C(O)C2=C1OC(C=1C=CC(O)=CC=1)=CC2=O SGEWCQFRYRRZDC-VPRICQMDSA-N 0.000 claims description 16
- PZKISQRTNNHUGF-UHFFFAOYSA-N vitexine Natural products OC1C(O)C(O)C(CO)OC1OC1=C(O)C=C(O)C2=C1OC(C=1C=CC(O)=CC=1)=CC2=O PZKISQRTNNHUGF-UHFFFAOYSA-N 0.000 claims description 16
- PCWAOEHIAAYHJX-WTLMAMESSA-N O([C@@H]1[C@H](C=23)O[C@@H]([C@H]([C@@H]1O)O)CO)C3=CC(O)=C(C(C=1)=O)C=2OC=1C1=CC=C(O)C=C1 Chemical compound O([C@@H]1[C@H](C=23)O[C@@H]([C@H]([C@@H]1O)O)CO)C3=CC(O)=C(C(C=1)=O)C=2OC=1C1=CC=C(O)C=C1 PCWAOEHIAAYHJX-WTLMAMESSA-N 0.000 claims description 15
- PCWAOEHIAAYHJX-UHFFFAOYSA-N chafuroside B Natural products OC1C(O)C(CO)OC(C=23)C1OC3=CC(O)=C(C(C=1)=O)C=2OC=1C1=CC=C(O)C=C1 PCWAOEHIAAYHJX-UHFFFAOYSA-N 0.000 claims description 15
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- 238000000354 decomposition reaction Methods 0.000 description 21
- BQEMYRDVBBRALV-STSJCENXSA-N [(2s,3r,4s,5s,6r)-2-[5,7-dihydroxy-2-(4-hydroxyphenyl)-4-oxochromen-6-yl]-4,5-dihydroxy-6-(hydroxymethyl)oxan-3-yl] hydrogen sulfate Chemical compound OS(=O)(=O)O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1C1=C(O)C=C(OC(=CC2=O)C=3C=CC(O)=CC=3)C2=C1O BQEMYRDVBBRALV-STSJCENXSA-N 0.000 description 20
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- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 14
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 12
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- OSALNYNYADJXIW-BOCLWZTGSA-N [(2s,3r,4s,5s,6r)-2-[5,7-dihydroxy-2-(4-hydroxyphenyl)-4-oxochromen-8-yl]-4,5-dihydroxy-6-(hydroxymethyl)oxan-3-yl] hydrogen sulfate Chemical compound OS(=O)(=O)O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1C1=C(O)C=C(O)C2=C1OC(C=1C=CC(O)=CC=1)=CC2=O OSALNYNYADJXIW-BOCLWZTGSA-N 0.000 description 8
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- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
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- 239000012153 distilled water Substances 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
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- 208000005623 Carcinogenesis Diseases 0.000 description 4
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- 239000007858 starting material Substances 0.000 description 4
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 3
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- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 3
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- VLSDXINSOMDCBK-UHFFFAOYSA-N 1,1'-azobis(N,N-dimethylformamide) Chemical compound CN(C)C(=O)N=NC(=O)N(C)C VLSDXINSOMDCBK-UHFFFAOYSA-N 0.000 description 2
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 description 2
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- RGHNJXZEOKUKBD-SQOUGZDYSA-N D-gluconic acid Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C(O)=O RGHNJXZEOKUKBD-SQOUGZDYSA-N 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- JPYHHZQJCSQRJY-UHFFFAOYSA-N Phloroglucinol Natural products CCC=CCC=CCC=CCC=CCCCCC(=O)C1=C(O)C=C(O)C=C1O JPYHHZQJCSQRJY-UHFFFAOYSA-N 0.000 description 2
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- ZTHYODDOHIVTJV-UHFFFAOYSA-N Propyl gallate Chemical compound CCCOC(=O)C1=CC(O)=C(O)C(O)=C1 ZTHYODDOHIVTJV-UHFFFAOYSA-N 0.000 description 2
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 description 2
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- 150000007513 acids Chemical class 0.000 description 2
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- 239000001099 ammonium carbonate Substances 0.000 description 2
- 239000000010 aprotic solvent Substances 0.000 description 2
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- CHHHXKFHOYLYRE-UHFFFAOYSA-M 2,4-Hexadienoic acid, potassium salt (1:1), (2E,4E)- Chemical compound [K+].CC=CC=CC([O-])=O CHHHXKFHOYLYRE-UHFFFAOYSA-M 0.000 description 1
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- 235000010331 calcium propionate Nutrition 0.000 description 1
- NEEHYRZPVYRGPP-UHFFFAOYSA-L calcium;2,3,4,5,6-pentahydroxyhexanoate Chemical compound [Ca+2].OCC(O)C(O)C(O)C(O)C([O-])=O.OCC(O)C(O)C(O)C(O)C([O-])=O NEEHYRZPVYRGPP-UHFFFAOYSA-L 0.000 description 1
- VEJCUEBBRSCJRP-UHFFFAOYSA-L calcium;hydron;phosphonato phosphate Chemical compound [Ca+2].OP(O)(=O)OP([O-])([O-])=O VEJCUEBBRSCJRP-UHFFFAOYSA-L 0.000 description 1
- 239000011545 carbonate/bicarbonate buffer Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- SIHHLZPXQLFPMC-UHFFFAOYSA-N chloroform;methanol;hydrate Chemical compound O.OC.ClC(Cl)Cl SIHHLZPXQLFPMC-UHFFFAOYSA-N 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
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- 229940061632 dehydroacetic acid Drugs 0.000 description 1
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- JEQRBTDTEKWZBW-UHFFFAOYSA-N dehydroacetic acid Chemical compound CC(=O)C1=C(O)OC(C)=CC1=O JEQRBTDTEKWZBW-UHFFFAOYSA-N 0.000 description 1
- 208000002925 dental caries Diseases 0.000 description 1
- 238000004332 deodorization Methods 0.000 description 1
- 238000010511 deprotection reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 description 1
- 235000019838 diammonium phosphate Nutrition 0.000 description 1
- 229910000388 diammonium phosphate Inorganic materials 0.000 description 1
- 235000019700 dicalcium phosphate Nutrition 0.000 description 1
- 235000015872 dietary supplement Nutrition 0.000 description 1
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 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
- XMXOIHIZTOVVFB-JIZZDEOASA-L disodium;(2s)-2-aminobutanedioate Chemical compound [Na+].[Na+].[O-]C(=O)[C@@H](N)CC([O-])=O XMXOIHIZTOVVFB-JIZZDEOASA-L 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000004318 erythorbic acid Substances 0.000 description 1
- 235000010350 erythorbic acid Nutrition 0.000 description 1
- BEFDCLMNVWHSGT-UHFFFAOYSA-N ethenylcyclopentane Chemical compound C=CC1CCCC1 BEFDCLMNVWHSGT-UHFFFAOYSA-N 0.000 description 1
- 238000004880 explosion Methods 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 235000019225 fermented tea Nutrition 0.000 description 1
- 229930003944 flavone Natural products 0.000 description 1
- 235000011949 flavones Nutrition 0.000 description 1
- 239000001530 fumaric acid Substances 0.000 description 1
- 229960002598 fumaric acid Drugs 0.000 description 1
- 238000007429 general method Methods 0.000 description 1
- 239000000174 gluconic acid Substances 0.000 description 1
- 235000012208 gluconic acid Nutrition 0.000 description 1
- 229950006191 gluconic acid Drugs 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- 150000002338 glycosides Chemical class 0.000 description 1
- 235000013402 health food Nutrition 0.000 description 1
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- 239000003999 initiator Substances 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- NPFOYSMITVOQOS-UHFFFAOYSA-K iron(III) citrate Chemical compound [Fe+3].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NPFOYSMITVOQOS-UHFFFAOYSA-K 0.000 description 1
- 229940026239 isoascorbic acid Drugs 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 229960000448 lactic acid Drugs 0.000 description 1
- ZLNQQNXFFQJAID-UHFFFAOYSA-L magnesium carbonate Chemical compound [Mg+2].[O-]C([O-])=O ZLNQQNXFFQJAID-UHFFFAOYSA-L 0.000 description 1
- 239000001095 magnesium carbonate Substances 0.000 description 1
- 229910000021 magnesium carbonate Inorganic materials 0.000 description 1
- BJEPYKJPYRNKOW-UHFFFAOYSA-N malic acid Chemical compound OC(=O)C(O)CC(O)=O BJEPYKJPYRNKOW-UHFFFAOYSA-N 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 239000012046 mixed solvent Substances 0.000 description 1
- 235000019691 monocalcium phosphate Nutrition 0.000 description 1
- 229930014626 natural product Natural products 0.000 description 1
- 239000008239 natural water Substances 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- WRMXOVHLRUVREB-UHFFFAOYSA-N phosphono phosphate;tributylazanium Chemical compound OP(O)(=O)OP([O-])([O-])=O.CCCC[NH+](CCCC)CCCC.CCCC[NH+](CCCC)CCCC WRMXOVHLRUVREB-UHFFFAOYSA-N 0.000 description 1
- 235000011007 phosphoric acid Nutrition 0.000 description 1
- AVTYONGGKAJVTE-OLXYHTOASA-L potassium L-tartrate Chemical compound [K+].[K+].[O-]C(=O)[C@H](O)[C@@H](O)C([O-])=O AVTYONGGKAJVTE-OLXYHTOASA-L 0.000 description 1
- 229910000027 potassium carbonate Inorganic materials 0.000 description 1
- OQZCJRJRGMMSGK-UHFFFAOYSA-M potassium metaphosphate Chemical compound [K+].[O-]P(=O)=O OQZCJRJRGMMSGK-UHFFFAOYSA-M 0.000 description 1
- 229940099402 potassium metaphosphate Drugs 0.000 description 1
- 239000004302 potassium sorbate Substances 0.000 description 1
- 235000010241 potassium sorbate Nutrition 0.000 description 1
- 229940069338 potassium sorbate Drugs 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 description 1
- 239000000473 propyl gallate Substances 0.000 description 1
- 235000010388 propyl gallate Nutrition 0.000 description 1
- 229940075579 propyl gallate Drugs 0.000 description 1
- 238000000197 pyrolysis Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- HELHAJAZNSDZJO-OLXYHTOASA-L sodium L-tartrate Chemical compound [Na+].[Na+].[O-]C(=O)[C@H](O)[C@@H](O)C([O-])=O HELHAJAZNSDZJO-OLXYHTOASA-L 0.000 description 1
- WXMKPNITSTVMEF-UHFFFAOYSA-M sodium benzoate Chemical compound [Na+].[O-]C(=O)C1=CC=CC=C1 WXMKPNITSTVMEF-UHFFFAOYSA-M 0.000 description 1
- 239000004299 sodium benzoate Substances 0.000 description 1
- 235000010234 sodium benzoate Nutrition 0.000 description 1
- 229960003885 sodium benzoate Drugs 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 235000019259 sodium dehydroacetate Nutrition 0.000 description 1
- 229940079839 sodium dehydroacetate Drugs 0.000 description 1
- FQENQNTWSFEDLI-UHFFFAOYSA-J sodium diphosphate Chemical compound [Na+].[Na+].[Na+].[Na+].[O-]P([O-])(=O)OP([O-])([O-])=O FQENQNTWSFEDLI-UHFFFAOYSA-J 0.000 description 1
- 239000001540 sodium lactate Substances 0.000 description 1
- 235000011088 sodium lactate Nutrition 0.000 description 1
- 229940005581 sodium lactate Drugs 0.000 description 1
- 239000001394 sodium malate Substances 0.000 description 1
- 235000019983 sodium metaphosphate Nutrition 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 235000011008 sodium phosphates Nutrition 0.000 description 1
- JXKPEJDQGNYQSM-UHFFFAOYSA-M sodium propionate Chemical compound [Na+].CCC([O-])=O JXKPEJDQGNYQSM-UHFFFAOYSA-M 0.000 description 1
- 239000004324 sodium propionate Substances 0.000 description 1
- 235000010334 sodium propionate Nutrition 0.000 description 1
- 229960003212 sodium propionate Drugs 0.000 description 1
- ZDQYSKICYIVCPN-UHFFFAOYSA-L sodium succinate (anhydrous) Chemical compound [Na+].[Na+].[O-]C(=O)CCC([O-])=O ZDQYSKICYIVCPN-UHFFFAOYSA-L 0.000 description 1
- PPASLZSBLFJQEF-RXSVEWSESA-M sodium-L-ascorbate Chemical compound [Na+].OC[C@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RXSVEWSESA-M 0.000 description 1
- 239000011755 sodium-L-ascorbate Substances 0.000 description 1
- 235000019187 sodium-L-ascorbate Nutrition 0.000 description 1
- DSOWAKKSGYUMTF-GZOLSCHFSA-M sodium;(1e)-1-(6-methyl-2,4-dioxopyran-3-ylidene)ethanolate Chemical compound [Na+].C\C([O-])=C1/C(=O)OC(C)=CC1=O DSOWAKKSGYUMTF-GZOLSCHFSA-M 0.000 description 1
- CIJQGPVMMRXSQW-UHFFFAOYSA-M sodium;2-aminoacetic acid;hydroxide Chemical compound O.[Na+].NCC([O-])=O CIJQGPVMMRXSQW-UHFFFAOYSA-M 0.000 description 1
- HBNPFAVGROHRSG-UHFFFAOYSA-M sodium;butanedioic acid;acetate Chemical compound [Na+].CC([O-])=O.OC(=O)CCC(O)=O HBNPFAVGROHRSG-UHFFFAOYSA-M 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 239000004334 sorbic acid Substances 0.000 description 1
- 235000010199 sorbic acid Nutrition 0.000 description 1
- 229940075582 sorbic acid Drugs 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- RYCLIXPGLDDLTM-UHFFFAOYSA-J tetrapotassium;phosphonato phosphate Chemical compound [K+].[K+].[K+].[K+].[O-]P([O-])(=O)OP([O-])([O-])=O RYCLIXPGLDDLTM-UHFFFAOYSA-J 0.000 description 1
- 235000019818 tetrasodium diphosphate Nutrition 0.000 description 1
- JHYAVWJELFKHLM-UHFFFAOYSA-H tetrasodium;2-hydroxypropane-1,2,3-tricarboxylate;iron(2+) Chemical compound [Na+].[Na+].[Na+].[Na+].[Fe+2].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O JHYAVWJELFKHLM-UHFFFAOYSA-H 0.000 description 1
- 238000005979 thermal decomposition reaction Methods 0.000 description 1
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 235000013337 tricalcium citrate Nutrition 0.000 description 1
- 229910000391 tricalcium phosphate Inorganic materials 0.000 description 1
- 235000019731 tricalcium phosphate Nutrition 0.000 description 1
- 229940078499 tricalcium phosphate Drugs 0.000 description 1
- 235000019798 tripotassium phosphate Nutrition 0.000 description 1
- 229910000404 tripotassium phosphate Inorganic materials 0.000 description 1
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 1
- 229940038773 trisodium citrate Drugs 0.000 description 1
- 235000019263 trisodium citrate Nutrition 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- VHBFFQKBGNRLFZ-UHFFFAOYSA-N vitamin p Natural products O1C2=CC=CC=C2C(=O)C=C1C1=CC=CC=C1 VHBFFQKBGNRLFZ-UHFFFAOYSA-N 0.000 description 1
Abstract
Description
本発明は、チャフロサイド類の簡易製造方法、特にチャフロサイド類の水溶液を対象とする処理方法の改善に係る。 The present invention relates to a simple method for producing chafurosides, and more particularly to an improvement of a treatment method for an aqueous solution of chafurosides.
チャフロサイド（ｃｈａｆｕｒｏｓｉｄｅ）は、天然に微量にしか存在せず、故に近年になって初めてウーロン茶から単離され、構造決定された天然化合物である（特許文献１）。それらは次に示す構造式を有し、フラボン誘導体の一種であるフラボンＣ配糖体に分類され、各々、チャフロサイドＡ、チャフロサイドＢと命名されている。 Chafuroside is a natural compound that is naturally present only in a trace amount, and therefore has been isolated from oolong tea for the first time in recent years and whose structure has been determined (Patent Document 1). They have the structural formula shown below, and are classified into flavone C glycosides, which are a kind of flavone derivative, and are named chafloside A and chafloside B, respectively.
チャフロサイドの効果作用としては、上記単離構造決定時の指標として用いられた抗アレルギー作用、あるいは発癌抑制作用（特許文献２）などが知られている。一方、茶類は、その製造工程中の発酵度合いにより主に緑茶に代表される不発酵茶、ウーロン茶に代表される半発酵茶、紅茶に代表される完全発酵茶の３種類に大別され、緑茶の代表的効能としては、発ガン抑制、抗酸化、抗菌（抗ウイルス）、覚醒、血圧・血糖値降下、消臭、虫歯予防が知られている。ウーロン茶の作用としては、抗酸化作用、抗アレルギー作用、抗炎症作用、αグルコシダーゼ( a l p h a - g l u c o s i d a s e )阻害作用、グルコシルトランスフェラーゼ( g l u c o s y l t r a n s f e r a s e )阻害作用等が知られている。 As an effective action of chafuroside, an antiallergic action used as an index at the time of determining the isolated structure or a carcinogenesis suppressing action (Patent Document 2) is known. On the other hand, teas are roughly classified into three types according to the degree of fermentation during the production process: unfermented tea typified by green tea, semi-fermented tea typified by oolong tea, and fully fermented tea typified by black tea. As representative effects of green tea, carcinogenesis suppression, anti-oxidation, antibacterial (antiviral), arousal, blood pressure / blood glucose level lowering, deodorization, and tooth decay prevention are known. As the action of oolong tea, an antioxidant action, an anti-allergic action, an anti-inflammatory action, an α-glucosidase (alphpha-glucosidase) inhibitory action, a glucosyltransferase (glucosyrsrase) inhibitory action and the like are known.
しかしながら、茶葉中のチャフロサイドの茶葉抽出物（Ｏｏｌｏｎｇ ｔｅａ ａｃｔｉｖｅ ｃｏｍｐｏｕｎｄ：ＯＴＡＣ）としての含有量は、ＨＰＬＣ分析法によれば、ウーロン茶で一番高く２４．８μｇ／ｇ（茶葉）であり、緑茶で８０ｎｇ／ｇ（茶葉）、焙じ茶で２．４μｇ／ｇ（茶葉）、紅茶で８０ｎｇ／ｇ（茶葉）程度であることが開示されている（特許文献２）。あるいは、特許文献３には、チャフロサイドＡとＢは茶間に大きな差が認められ、緑茶及び紅茶中の含量は茶葉１グラム当たり数１０ｎｇで、銘柄間の差はほとんどなく、焙じ茶とウーロン茶ではバラツキはあるが含量は茶葉１グラム当たり数μｇで、焙じ茶では銘柄間には有意な差はなく、ウーロン茶では大きな差があった旨が記載されている（同段落番号００８３）。 However, the content of chafloside in tea leaves as Oolong tea active compound (OTAC) is the highest in oolong tea, 24.8 μg / g (tea leaves) according to HPLC analysis, and 80 ng in green tea / G (tea leaves), roasted tea is 2.4 μg / g (tea leaves), and black tea is about 80 ng / g (tea leaves) (Patent Document 2). Alternatively, in Patent Document 3, there is a large difference between Chafroside A and B, the content in green tea and black tea is several tens of ng per gram of tea leaves, there is almost no difference between brands, and variation between roasted tea and oolong tea. However, the content is several μg per gram of tea leaves, and it is described that there is no significant difference between brands in roasted tea and there is a large difference in oolong tea (same paragraph number 0083).
したがって、チャフロサイドの更なる効果作用の解明が待たれるが、ウーロン茶中においてさえ、その含有量が少なく、その精製も面倒なため、その量の確保が大きな問題となっている。 Therefore, elucidation of further effective action of chafuroside is awaited, but even in oolong tea, its content is small and its purification is troublesome, so securing the amount is a big problem.
その解決法として、ハーブなどに含まれるイソビテキシンおよびビテキシンを原料として、１、１’−アゾビス（Ｎ、Ｎ’−ジメチルホルムアミド）、トリ−ｎ−ブチルホスフィンを用いたチャフロサイドＡ及びＢの合成法（特許文献４）が開示されているが、本方法は爆発の危険の高いアゾ試薬を用いるためにその工業化は極めて難しい状況にある。 As a solution to this problem, a method for synthesizing chafuroside A and B using 1,1′-azobis (N, N′-dimethylformamide) and tri-n-butylphosphine using isovitexin and vitexin contained in herbs as raw materials ( Although Patent Document 4) is disclosed, since this method uses an azo reagent having a high risk of explosion, its industrialization is extremely difficult.
また、非プロトン性溶媒中で、ルイス酸触媒の存在下で、ベンゼン環に糖類を結合させる第１段階、非プロトン性溶媒中で、糖とベンゼン環の結合化合物をアゾジカルボン酸アミド等（例えば１、１’−アゾビス（Ｎ、Ｎ’−ジメチルホルムアミド）を用いる上記特許文献４記載の方法）と反応させる第２段階を経るチャフロサイドＡの合成法（特許文献５）が報告されている。具体的には、１、３、５−ベンゼントリオールを出発原料として３工程で合成されるベンゼン環化合物に、ルイス酸触媒の存在下で、糖類を結合させて糖とベンゼン環の結合化合物を得る（ここまでの収率が３．３％）。次いで、本結合化合物を３工程の反応に付してイソビテキシンの保護体を得、さらに、１、１’−アゾビス（Ｎ、Ｎ’−ジメチルホルムアミド）、トリ−ｎ−ブチルホスフィンを用いて縮環、次いで脱保護反応することにより目的のチャフロサイドＡを得る。１、３、５−ベンゼントリオールからの全収率は０．１０％に過ぎず、かなり高価な合成法であり、かつ、アゾ試薬を用いることによる危険性を伴う点では特許文献４の場合と同じである。 Further, in the first step of bonding a saccharide to a benzene ring in the presence of a Lewis acid catalyst in an aprotic solvent, an azodicarboxylic acid amide or the like (for example, a compound binding a sugar and a benzene ring in an aprotic solvent) A method for synthesizing chafuroside A (Patent Document 5), which undergoes a second step of reacting with 1,1′-azobis (the method described in Patent Document 4 using N, N′-dimethylformamide), has been reported. Specifically, a saccharide is bonded to a benzene ring compound synthesized in three steps using 1,3,5-benzenetriol as a starting material in the presence of a Lewis acid catalyst to obtain a combined compound of sugar and benzene ring. (The yield so far is 3.3%). Subsequently, this binding compound is subjected to a three-step reaction to obtain a protected product of isovitexin, and further condensed with 1,1′-azobis (N, N′-dimethylformamide) and tri-n-butylphosphine. Then, the desired chafuroside A is obtained by deprotection reaction. The total yield from 1,3,5-benzenetriol is only 0.10%, which is a fairly expensive synthesis method and has a risk due to the use of an azo reagent. The same.
そこで、チャフロサイドの生合成前駆体であるイソビテキシンおよびビテキシンの硫酸化体を用いてチャフロサイドを製造しようとする試みがなされている（特許文献３）。イソビテキシンおよびビテキシンの硫酸化体（イソビテキシン２”−サルフェイト、ビテキシン２”−サルフェイト）としては、茶葉又は茶渋から抽出・単離したものを用いてもよいし、あるいは、天然から単離したイソビテキシン又はビテキシンから次のようにして合成して用いてもよい。すなわち、イソビテキシン又はビテキシンの４位と５位の水酸基を予め保護し、次いで、三酸化イオウ−ピリジン錯体などを用いて所望の２位の水酸基を硫酸化し、最後に、保護基を外し、目的とする硫酸化体を得ることができる。このようにして得られた各硫酸化体を、１３０〜１９０℃で加熱することにより、チャフロサイドＡ又はＢを製造できることが特許文献３に記載されている。具体的には、同段落番号０１１７、０１２２に、チャフロサイドＡ又はＢの前駆体を約１６０〜１７０℃で加熱したところチャフロサイドＡ又はＢを収率約８５％で得たと記載されている。 Thus, attempts have been made to produce chafuroside using isovitexin, which is a biosynthetic precursor of chafuroside, and a sulfated form of vitexin (Patent Document 3). As isovitexin and sulfated form of vitexin (isovitexin 2 "-sulfate, vitexin 2" -sulfate), those extracted and isolated from tea leaves or tea astringents may be used, or isovitexin isolated from nature Alternatively, it may be synthesized from vitexin as follows. Specifically, isovitexin or the hydroxyl groups at positions 4 and 5 of vitexin are protected in advance, then the desired hydroxyl group at the 2-position is sulfated using a sulfur trioxide-pyridine complex, and finally the protecting group is removed, To obtain a sulfated product. Patent Document 3 describes that chafloside A or B can be produced by heating each sulfated product thus obtained at 130 to 190 ° C. Specifically, it is described in the paragraph Nos. 0117 and 0122 that when the precursor of chafuroside A or B was heated at about 160 to 170 ° C., chafloside A or B was obtained at a yield of about 85%.
しかしながら、特許文献３中の記載からは、該加熱は、前駆体に対し何らかの添加物を加える、あるいは、何らかの溶媒を用いて溶液状態で加熱を行ったのものではなく、前駆体をそのままｎｅａｔ状態で加熱したものであると判断できる。粘着性ｎｅａｔ状の貴重な前駆体をロスすることなく反応釜に移すのはかなり面倒であり、ハンドリング上の不都合を避けることは出来ない。また、チャフロサイドは１６０℃以上では少しずつ分解することが知られており、該加熱時間も定かでないことから、高温かつ長時間の熱処理を行うことにより、生じたチャフロサイドの分解も懸念される。更には、実製造の面でも、このように１５０℃以上の加熱のためには、通常用いる加圧水蒸気では不十分であり、加圧水蒸気に代わる新たな加熱設備が必要となる。このような点に鑑み、ハンドリングが容易で、より一層低い温度での変換反応が可能な、経済性、実用性に富む製造方法へのより一層の改良が求められている。
本発明においては、上記のような操作性面と高温加熱処理に伴う欠点を改善し、実製造面での操作性に優れ、比較的低温かつ短時間の加熱処理によって、チャフロサイド類を効率よく簡便に製造する方法を提供することを目的とするものである
However, from the description in Patent Document 3, the heating is not performed by adding any additive to the precursor, or by heating in a solution state using any solvent, and the precursor is left in the neat state as it is. It can be determined that the product has been heated. It is quite troublesome to transfer the precious precursor in the form of sticky neat to the reaction kettle without loss, and inconvenience in handling cannot be avoided. In addition, it is known that chafloside is decomposed little by little at 160 ° C. or more, and the heating time is not clear. Furthermore, from the viewpoint of actual production, the pressurized steam used normally is not sufficient for heating at 150 ° C. or higher as described above, and a new heating facility is required instead of the pressurized steam. In view of these points, there is a need for further improvements to a manufacturing method that is easy to handle and can perform a conversion reaction at a lower temperature and is rich in economy and practicality.
In the present invention, the above-described operability and disadvantages associated with high-temperature heat treatment are improved, operability in actual production is excellent, and chafurosides are efficiently and easily produced by relatively low-temperature and short-time heat treatment. The purpose is to provide a manufacturing method
上記事情に鑑み、本発明者は、操作性を考慮して水溶液での反応を試みたが、事実上反応は進行しなかった。そこで、鋭意検討した結果、酸、塩基、塩のいずれか、若しくはそれらの混合物を反応水溶液に加えるこることにより、反応が進行しやすくなることを見出した。すなわち、チャフロサイド類の前駆体である硫酸化体を高温で直接加熱して、チャフロサイド類を製造する方法ではなく、チャフロサイド類の前駆体を水に溶解して水溶液とし、酸、塩基、塩のいずれか、若しくはそれらの混合物の存在下、ｐＨ５〜１２の範囲において、１００〜１５０℃で加熱処理することにより、チャフロサイド類を操作性よく、効率的かつ簡便に製造する方法を明らかにし、本発明を完成するに至った。 In view of the above circumstances, the present inventor attempted a reaction in an aqueous solution in consideration of operability, but the reaction did not proceed in practice. Thus, as a result of intensive studies, it was found that the reaction can easily proceed by adding any of acid, base, salt, or a mixture thereof to the reaction aqueous solution. That is, it is not a method in which a sulfated body, which is a precursor of chaflosides, is directly heated at a high temperature to produce chaflosides, but the precursor of chaflosides is dissolved in water to form an aqueous solution, and any of acid, base, and salt Or in the presence of a mixture thereof, in the range of pH 5 to 12, by heating at 100 to 150 ° C., a method for producing chafurosides with good operability, efficiency and simplicity is clarified. It came to be completed.
すなわち、本発明は、チャフロサイド類の前駆体である硫酸化体から、チャフロサイド類を製造する方法で、前記製造方法が、以下の工程（１）〜（３）を含むことを特徴とする製造方法である。
（１）前記硫酸化体を下記の加熱工程に先立ち、酸、塩基、塩のいずれか、若しくはそれらの混合物を含有する水溶液とする工程、
（２）前記工程（１）において、下記加熱工程に付す水溶液のｐＨを５〜１２に調整する工程、および
（３）前記工程（２）を経て得られるｐＨ調整済水溶液を、１００〜１５０℃で加熱処理する工程。
That is, the present invention is a method for producing chafurosides from a sulfated body that is a precursor of chafrosides, wherein the production method comprises the following steps (1) to (3): It is.
(1) Prior to the following heating step, the sulfated body is an aqueous solution containing any one of an acid, a base, a salt, or a mixture thereof,
(2) In the step (1), the step of adjusting the pH of the aqueous solution to be subjected to the following heating step to 5 to 12, and (3) the pH-adjusted aqueous solution obtained through the step (2) is 100 to 150 ° C. The process of heat-treating with.
本発明にかかるチャフロサイド類の製造方法は、前記チャフロサイド類の前駆体である硫酸化体が、イソビテキシン２”−サルフェイト及び／又はビテキシン２”−サルフェイトであることを特徴とするチャフロサイド類の製造方法である。 The method for producing chafurosides according to the present invention is characterized in that the sulfated precursor of the chafurosides is isovitexin 2 ″ -sulfate and / or vitexin 2 ″ -sulfate. Is the method.
本発明にかかるチャフロサイド類の製造方法は、前記チャフロサイド類が、チャフロサイドＡ及び／又はチャフロサイドＢであることを特徴とするチャフロサイド類の製造方法である。 The method for producing chaflosides according to the present invention is a method for producing chaflosides, wherein the chaflosides are chafloside A and / or chafloside B.
本発明にかかるチャフロサイド類の製造方法は、前記工程（１）で用いられる酸、塩基、塩の混合物が、ｐＨ緩衝液であることを特徴とするチャフロサイド類の製造方法である。 The method for producing chaflosides according to the present invention is a method for producing chaflosides, wherein the mixture of acid, base and salt used in the step (1) is a pH buffer solution.
本発明にかかるチャフロサイド類の製造方法は、前記工程（１）で用いられる酸、塩基、塩のいずれかの量、若しくはそれらの混合物の合計量が、ｐＨ調整済水溶液中のチャフロサイド類の前駆体である硫酸化体に対し塩基性物質として作用する量として、１当量以上であることを特徴とするチャフロサイド類の製造方法である。 In the method for producing chaflosides according to the present invention, the amount of any one of the acid, base, and salt used in the step (1), or the total amount of the mixture is a precursor of chafuroside in the pH-adjusted aqueous solution. It is a manufacturing method of the chafuroside characterized by being 1 equivalent or more as an amount which acts as a basic substance with respect to the sulfated substance which is.
本発明にかかるチャフロサイド類の製造方法は、前記工程（３）における加熱処理が、加熱時間１分〜１６分であることを特徴とするチャフロサイド類の製造方法である。 The method for producing chaflosides according to the present invention is a method for producing chaflosides, wherein the heat treatment in the step (3) is performed for 1 to 16 minutes.
本発明にかかるチャフロサイド類の製造方法は、前記工程（３）における加熱処理が、マイクロウェーブ若しくは熱交換器を用いた加熱処理であることを特徴とするチャフロサイド類の製造方法である。 The method for producing chaflosides according to the present invention is a method for producing chaflosides, wherein the heat treatment in the step (3) is a heat treatment using a microwave or a heat exchanger.
本発明にかかるチャフロサイド類の製造方法は、前記チャフロサイド類の前駆体である硫酸化体が、茶葉の抽出物から得られた前記硫酸化体の濃縮混合物であることを特徴とするチャフロサイド類の製造方法である。 The method for producing chaflosides according to the present invention is characterized in that the sulfated body that is a precursor of the chaflosides is a concentrated mixture of the sulfated body obtained from an extract of tea leaves. Is the method.
さらには、本発明にかかるチャフロサイド類の製造方法は、前記工程（３）の加熱処理の後に得られた反応混合物を、さらに精製工程に付すことによってチャフロサイドＡ及び／又はチャフロサイドＢを得ることを特徴とするチャフロサイド類の製造方法である。 Furthermore, the method for producing chaflosides according to the present invention is characterized in that chafuroside A and / or chafuroside B is obtained by subjecting the reaction mixture obtained after the heat treatment in the step (3) to a purification step. It is a manufacturing method of chafuroside.
本発明のチャフロサイド類の製造方法は、高圧水蒸気に代表される簡易な加熱方法、あるいはマイクロウェーブを用いた加熱方法により、比較的低い温度で、かつ数１０分以内の加熱時間で、必要によってはバッチ処理ではなく連続的に、簡便かつ効率的にチャフロサイド類を製造することが出来る方法を提供出来る。 The production method of the chafurosides of the present invention can be carried out at a relatively low temperature and within a heating time of several tens of minutes by a simple heating method typified by high-pressure steam or a heating method using microwaves. Instead of batch processing, it is possible to provide a method capable of producing chafuroside continuously and simply and efficiently.
本発明の製造方法により、各種の目的に供するためのチャフロサイド類を、安価にかつ持続的に製造・提供すること可能になるので、これまで十分に解明されていなかったチャフロサイド類の有用な新たな用途を見出すことに利用できる。 The production method of the present invention makes it possible to produce and provide chafurosides for various purposes at a low cost and continuously. Therefore, useful new and useful chafurosides that have not been sufficiently elucidated so far. It can be used to find uses.
また、少なくとも現時点で明らかとなっている抗酸化作用、抗アレルギー作用、抗炎症作用、発癌抑制作用を有する化合物として、医薬品用途に供するチャフロサイド類を安価に製造することができる。 Further, as a compound having at least an antioxidant action, an antiallergic action, an anti-inflammatory action, and a carcinogenesis-suppressing action that are currently known, chafurosides for use in pharmaceuticals can be produced at low cost.
さらに、本発明のチャフロサイド類の製造方法で製造されたチャフロサイド類は、チャフロサイド類の生理活性に基づく、健康を維持するための種々の効果（予防的な効果）を有する製品を提供することが出来、特定保健用食品、特殊栄養食品、栄養補助食品として、あるいはその他の栄養食品、健康食品に利用することができる。 Furthermore, the chaflosides produced by the method for producing chaflosides of the present invention can provide products having various effects (preventive effects) for maintaining health based on the physiological activity of the chafurosides. It can be used as a food for specified health use, special nutritional food, dietary supplement, or other nutritional food and health food.
以下に、本発明の実施の形態について詳述する。
本発明におけるチャフロサイド類とは、少なくとも前記の化１で表されるチャフロイドＡ及びＢを意味する。
Hereinafter, embodiments of the present invention will be described in detail.
The chaflosides in the present invention mean at least the chafroids A and B represented by the above chemical formula 1.
本発明の出発原料であるチャフロサイド類の前駆体、すなわち硫酸化体（イソビテキシン２”−サルフェイト、ビテキシン２”−サルフェイト）は、前記特許文献３に記載されている方法によりイソビテキシンおよびビテキシンから製造することが出来る。イソビテキシンおよびビテキシン自体は、ハーブ、そば殻を始め多くの植物に含まれる成分であり、溶媒抽出および分離精製工程により得ることができる。植物の中には、イソビテキシンおよびビテキシンを高い割合で含有する植物も存在し、それらは容易に入手可能である。具体的には、例えば、Ｎｅｗ Ｚｅａｌａｎｄ産のＰｕｒｉｒｉの乾燥木部１ｋｇから、イソビテキシンおよびビテキシンを各２ｇ及び０．５ｇ得ることが出来る（未発表データ）。これらを原料として、先ず、イソビテキシン又はビテキシンの４位と５位の水酸基を予め保護し、次いで、三酸化イオウ−ピリジン錯体などを用いて所望の２位の水酸基を硫酸化する。最後に、保護基を外し、目的とするチャフロサイドＡ又はＢの前駆体である硫酸化体（イソビテキシン２”−サルフェイト、ビテキシン２”−サルフェイト）を得ることができる。 The precursor of chaflosides, ie, sulfated form (isovitexin 2 ″ -sulfate, vitexin 2 ″ -sulfate), which is the starting material of the present invention, is produced from isovitexin and vitexin by the method described in Patent Document 3. I can do it. Isovitexin and vitexin itself are components contained in many plants including herbs and buckwheat husks, and can be obtained by solvent extraction and separation and purification processes. Some plants contain a high proportion of isovitexin and vitexin, and they are readily available. Specifically, for example, 2 g and 0.5 g of isovitexin and vitexin can be obtained from 1 kg of dry xylem of Puriri from New Zealand (unpublished data). Using these as starting materials, first, the 4- and 5-position hydroxyl groups of isovitexin or vitexin are protected in advance, and then the desired 2-position hydroxyl group is sulfated using a sulfur trioxide-pyridine complex or the like. Finally, the protecting group is removed, and the sulfated form (isovitexin 2 ″ -sulfate, vitexin 2 ″ -sulfate), which is the target precursor of chafuroside A or B, can be obtained.
上記の硫酸化体は、単一な化合物であってもよいし、あるいは硫酸化体の混合物であってもよい。前記混合物としては、例えば、茶葉の抽出物を液液分配、カラムクロマトグラフィー等に付すことにより得られる硫酸化体含量の高い混合物を挙げることが出来る。該混合物を更に精製すれば、イソビテキシン２”−サルフェイト及びビテキシン２”−サルフェイトを各々得ることが出来る。単一な硫酸化体を用いて前記工程（１）〜（３）を行った場合でも、この様な混合物を用いて行った場合にも、前記工程（３）の後に、反応液をカラムクロマトグラフィー等の分離手段を用いて精製することにより、単一化合物としてのチャフロサイドを得ることが出来る。 The sulfated product may be a single compound or a mixture of sulfated products. Examples of the mixture include a mixture having a high sulfated content obtained by subjecting a tea leaf extract to liquid-liquid distribution, column chromatography, and the like. If the mixture is further purified, isovitexin 2 "-sulfate and vitexin 2" -sulfate can be obtained, respectively. Even when the steps (1) to (3) are performed using a single sulfate, or when such a mixture is used, the reaction solution is subjected to column chromatography after the step (3). By purifying using a separation means such as chromatography, chafuroside as a single compound can be obtained.
前記硫酸化体含量の高い混合物は、具体的には次のようにして得られるがこの方法に限定されるものではない。茶葉または茶渋から茶抽出液を調製する方法としては、原料茶葉、茶渋またはそれらの粉末を一般的な方法で抽出すればよい。例えば、抽出釜に茶葉を仕込んだ後に所定量の水あるいは水−アルコール混液で一定時間浸漬させ、次いで、茶殻及び不溶物を濾過もしくは遠心分離によって除去して抽出液を得る。抽出に使用する水としては、特に制限は無く、目的に応じて適宜選択することができる。アルコールとしては、炭素数が１〜３のメタノール、エタノール、プロパノールが挙げられる。抽出に使用する溶媒の量は、原料茶葉、茶渋またはそれらの粉末が十分に浸る量であれば特に限定されないが、通常、使用する原料茶葉、茶渋またはそれらの粉末の質量に対して５倍量以上が好ましい。抽出時の溶媒温度は、抽出できる温度であれば特に限定されないが、通常４〜９５℃程度であり、好ましくは３０〜９０℃である。抽出時間についても特に限定されないが、通常１分〜１２時間程度であり、好ましくは５分〜６時間である。 The mixture having a high sulfated content is specifically obtained as follows, but is not limited to this method. As a method for preparing a tea extract from tea leaves or tea astringents, raw material tea leaves, tea astringents or powders thereof may be extracted by a general method. For example, tea leaves are charged into an extraction kettle and immersed in a predetermined amount of water or a water-alcohol mixture for a certain period of time, and then the tea husk and insoluble matter are removed by filtration or centrifugation to obtain an extract. There is no restriction | limiting in particular as water used for extraction, According to the objective, it can select suitably. As alcohol, C1-C3 methanol, ethanol, and a propanol are mentioned. The amount of the solvent used for extraction is not particularly limited as long as the raw material tea leaves, tea astringents or their powders are sufficiently immersed, but usually 5 times the mass of the raw material tea leaves, tea astringents or their powders used. The above is preferable. Although the solvent temperature at the time of extraction will not be specifically limited if it is the temperature which can be extracted, Usually, it is about 4-95 degreeC, Preferably it is 30-90 degreeC. The extraction time is not particularly limited, but is usually about 1 minute to 12 hours, preferably 5 minutes to 6 hours.
次いで、前記茶抽出液を減圧下、好ましくは１０〜５００ｍｍＨｇの減圧下、適切な温度、すなわち、通常は用いた溶媒の沸点より１０〜３０℃高い温度において濃縮・乾固させ、実質的に溶媒を除去した茶抽出乾燥残分を得ることができる。さらには、前記茶抽出乾燥残分を、液液分配、カラムクロマトグラフィーに付し、チャフロサイド類の前駆体（イソビテキシン２”−サルフェイト、ビテキシン２”−サルフェイト）の濃縮混合物を得ることができる。 Next, the tea extract is concentrated and dried under a reduced pressure, preferably under a reduced pressure of 10 to 500 mmHg, at an appropriate temperature, that is, usually 10 to 30 ° C. higher than the boiling point of the solvent used. It is possible to obtain a tea extraction dry residue from which the odor is removed. Further, the tea extract dried residue can be subjected to liquid-liquid distribution and column chromatography to obtain a concentrated mixture of chafuroside precursors (isovitexin 2 ″ -sulfate, vitexin 2 ″ -sulfate). .
前記工程（１）において、チャフロサイド類の前駆体である硫酸化体を水に溶かして水溶液とするが、溶解に使用する水としては、特に制限は無く、目的に応じて適宜選択することができ、例えば、水道水、イオン交換水、蒸留水、純水、超純水、ナチュラルウォーター、ナチュラルミネラルウォーター、脱気水などを挙げることができる。 In the step (1), the sulfated body, which is a precursor of chafurosides, is dissolved in water to obtain an aqueous solution, but the water used for dissolution is not particularly limited and can be appropriately selected according to the purpose. Examples thereof include tap water, ion exchange water, distilled water, pure water, ultrapure water, natural water, natural mineral water, and deaerated water.
前記工程（１）において、チャフロサイド類の前駆体である硫酸化体の水溶液中の濃度は、特に制限が無く、加熱条件、特に、加熱装置の特性を最大限生かせるように設定すればよい。一般的には、高濃度の方が経済的価値は高く、製造単価を下げることが可能であるが、８０％以上の濃度においては反応水溶液が粘稠になりすぎ扱いにくくなる。好ましくは、前駆体の濃度は、０．０００１〜８０％、更に好ましくは、０．００１〜５０％、特に好ましくは、０．００１〜３０％である。 In the step (1), the concentration of the sulfated body, which is a precursor of the chafuroside, in the aqueous solution is not particularly limited, and may be set so that the heating conditions, particularly the characteristics of the heating apparatus, can be utilized to the maximum. In general, the higher the concentration, the higher the economic value and the lower the production unit price. However, when the concentration is 80% or more, the aqueous reaction solution becomes too viscous and difficult to handle. Preferably, the concentration of the precursor is 0.0001 to 80%, more preferably 0.001 to 50%, and particularly preferably 0.001 to 30%.
前記工程（１）で使用される酸、塩基、塩としては、特に制限は無く、目的に応じて適宜選択することが出来、例えば以下の酸、塩基、塩を挙げることが出来る。水酸化ナトリウム、炭酸アンモニウム、炭酸カリウム、炭酸カルシウム、炭酸水素アンモニウム、炭酸水素ナトリウム、炭酸ナトリウム、炭酸マグネシウム、ピロリン酸二水素カルシウム、ピロリン酸二水素ナトリウム、ピロリン酸四カリウム、ピロリン酸四ナトリウム、ポリリン酸ナトリウム、メタリン酸カリウム、メタリン酸ナトリウム、リン酸、リン酸一水素カルシウム、リン酸三カリウム、リン酸三カルシウム、リン酸水素二アンモニウム、リン酸水素二カリウム、リン酸水素二ナトリウム、リン酸三ナトリウム、リン酸二水素カルシウム、リン酸二水素ナトリウム、アジピン酸、Ｌ−アスコルビン酸、Ｌ−アスコルビン酸ナトリウム、Ｌ−アスパラギン酸ナトリウム、安息香酸、安息香酸ナトリウム、エリソルビン酸、エリソルビン酸ナトリウム、クエン酸、クエン酸カルシウム、クエン酸第一鉄ナトリウム、クエン酸鉄、クエン酸鉄アンモニウム、クエン酸三ナトリウム、グルコン酸、グルコン酸カルシウム、コハク酸、コハク酸二ナトリウム、酢酸、酢酸ナトリウム、蓚酸、Ｌ−酒石酸、Ｌ−酒石酸水素カリウム、Ｌ−酒石酸ナトリウム、ソルビン酸、ソルビン酸カリウム、デヒドロ酢酸、デヒドロ酢酸ナトリウム、乳酸、乳酸ナトリウム、パントテン酸カルシウム、フマル酸、フマル酸一ナトリウム、プロピオン酸、プロピオン酸カルシウム、プロピオン酸ナトリウム、没食子酸プロピル、ＤＬ−リンゴ酸、ＤＬ−リンゴ酸ナトリウム、グリシン、Ｌ−グルタミン酸ナトリウム。 There is no restriction | limiting in particular as an acid, a base, and a salt used by the said process (1), According to the objective, it can select suitably, For example, the following acids, bases, and salts can be mentioned. Sodium hydroxide, ammonium carbonate, potassium carbonate, calcium carbonate, ammonium bicarbonate, sodium bicarbonate, sodium carbonate, magnesium carbonate, calcium dihydrogen pyrophosphate, sodium dihydrogen pyrophosphate, tetrapotassium pyrophosphate, tetrasodium pyrophosphate, polyphosphorus Sodium phosphate, potassium metaphosphate, sodium metaphosphate, phosphoric acid, calcium monohydrogen phosphate, tripotassium phosphate, tricalcium phosphate, diammonium hydrogen phosphate, dipotassium hydrogen phosphate, disodium hydrogen phosphate, phosphoric acid Trisodium, calcium dihydrogen phosphate, sodium dihydrogen phosphate, adipic acid, L-ascorbic acid, sodium L-ascorbate, sodium L-aspartate, benzoic acid, sodium benzoate, erythorbic acid, erythorbium Acid sodium, citric acid, calcium citrate, sodium ferrous citrate, iron citrate, ammonium iron citrate, trisodium citrate, gluconic acid, calcium gluconate, succinic acid, disodium succinate, acetic acid, sodium acetate Succinic acid, L-tartaric acid, potassium L-tartrate, sodium L-tartrate, sorbic acid, potassium sorbate, dehydroacetic acid, sodium dehydroacetate, lactic acid, sodium lactate, calcium pantothenate, fumaric acid, monosodium fumarate, propion Acid, calcium propionate, sodium propionate, propyl gallate, DL-malic acid, DL-sodium malate, glycine, sodium L-glutamate.
前記工程（１）で使用される酸、塩基、塩は、それらの混合物として用いてもよく、その組み合わせに特に制限は無い。目的に応じて適宜選択することが出来るが、前記工程（３）で加熱処理をすることから、緩衝液を用いるのが好ましい。緩衝液としては、例えば、クエン酸緩衝液（ｐＨ３．０〜６．２）、酢酸緩衝液（ｐＨ３．６〜５．６）、クエン酸−リン酸緩衝液（ｐＨ２．６〜７．０）、リン酸緩衝液（ｐＨ５．８〜８．０）、グリシン−水酸化ナトリウム緩衝液（ｐＨ８．６〜１０．６）あるいは炭酸−重炭酸緩衝液（ｐＨ９．２〜１０．６）などを挙げることが出来る。 The acid, base and salt used in the step (1) may be used as a mixture thereof, and the combination thereof is not particularly limited. Although it can select suitably according to the objective, since it heat-processes at the said process (3), it is preferable to use a buffer solution. Examples of the buffer solution include a citrate buffer solution (pH 3.0 to 6.2), an acetate buffer solution (pH 3.6 to 5.6), and a citrate-phosphate buffer solution (pH 2.6 to 7.0). , Phosphate buffer (pH 5.8 to 8.0), glycine-sodium hydroxide buffer (pH 8.6 to 10.6), carbonate-bicarbonate buffer (pH 9.2 to 10.6), etc. I can do it.
前記工程（１）で使用される酸、塩基、塩のいずれか、若しくはそれらの混合物は、チャフロサイド類の前駆体である硫酸化体に対し塩基性物質として作用する量として、１当量以上使用する必要がある。使用される酸、塩基、塩は、チャフロサイド類の前駆体を溶解する際にｐＨ調整水（酸、塩基、塩のいずれか、若しくはそれらの混合物を含む）として用いられてもよいし、前記の水（蒸留水、イオン交換水等）を用いて該前駆体を溶解した後に添加されてもよい。 The acid, base, salt, or mixture thereof used in the step (1) is used in an amount of 1 equivalent or more as an amount that acts as a basic substance for the sulfated form that is a precursor of chafurosides. There is a need. The acid, base, or salt used may be used as pH-adjusted water (including any of acid, base, salt, or a mixture thereof) when dissolving the precursor of chafuroside. The precursor may be added after dissolving the precursor using water (distilled water, ion-exchanged water, etc.).
前記工程（２）においては、前記工程（３）の加熱処理に供する水溶液のｐＨを、５〜１２の範囲で所望の値に調整し、ｐＨ調整済水溶液を得る。概して、前記工程（３）の加熱処理においては、ｐＨ値が高くなるとチャフロサイド類の生成速度が上がり、短時間でチャフロサイド類の含有濃度を上げることが出来るが、加熱装置や加熱処理量等の要件に応じてｐＨを設定すればよい。ｐＨ値が５未満の強酸性条件では、前記工程（３）の加熱処理を行っても、チャフロサイド類の生成は認められず、前駆体から硫酸基が脱離したイソビテキシン、あるいはビテキシンが生じてしまう。一方、ｐＨ１２よりも強い塩基性条件においては、比較的低い加熱温度においてもチャフロサイド類の分解が極めて起こりやすくなり、また、その強い塩基性故に扱いに困難さが生じ、かつ、反応処理時に多量の酸を用いて中和する必要があるなど、多くの問題が生じることから、反応条件としては好ましくない。 In the step (2), the pH of the aqueous solution subjected to the heat treatment in the step (3) is adjusted to a desired value in the range of 5 to 12 to obtain a pH adjusted aqueous solution. In general, in the heat treatment of the step (3), when the pH value is increased, the generation rate of chafurosides increases, and the content concentration of chafurosides can be increased in a short time. The pH may be set according to the conditions. Under strongly acidic conditions with a pH value of less than 5, even if the heat treatment in the above step (3) is performed, the formation of chafurosides is not recognized, and isovitexin or vitexin from which the sulfate group has been eliminated from the precursor is generated. . On the other hand, in basic conditions stronger than pH 12, the decomposition of chafurosides is extremely likely to occur even at a relatively low heating temperature, and it is difficult to handle due to its strong basicity. Since many problems arise, such as the need to neutralize with an acid, the reaction conditions are not preferred.
前記工程（１）及び（２）を行わない場合、すなわち、酸、塩基、塩のいずれか、若しくはそれらの混合物を用いることなく、又は用いたとしても所定のｐＨ範囲に収まるようにｐＨ調整を行うことなく、次の加熱工程（３）に付した場合には、本発明の目的とするチャフロサイド類を高収率で得ることは難しくなる。 In the case where the steps (1) and (2) are not performed, that is, without any acid, base, salt, or a mixture thereof, or even if used, the pH is adjusted so as to be within a predetermined pH range. Without carrying out, when it puts on the following heating process (3), it will become difficult to obtain the chafuroside which is the objective of this invention with a high yield.
前記工程（３）における加熱方法は特に限定されない。例えば、加圧水蒸気を用いた加熱釜（反応釜）、あるいは熱交換装置（プレート式熱交換器、スパイラル式熱交換器等）等による単純加熱であっても、マイクロウェーブを用いた加熱であっても良い。熱交換装置においては、加熱時間を短くするためにできるだけ熱交換表面積が広いものが好ましい。例えば、スパイラル式導管内へ前記ｐＨ調整済の前駆体水溶液を、流量を制御しながら通導し、導管周囲を加圧水蒸気で一定温度に加熱することにより、加熱時間を一定に保ちながらの連続合成が可能となる。また、マイクロウェーブを用いる場合には、その照射条件は特に制限されないが、例えば、２４５０±３０ＭＨｚのマイクロ波を、好ましくは３０Ｗ以上、さらに好ましくは１００〜４００Ｗの出力で照射するのが好ましい。 The heating method in the step (3) is not particularly limited. For example, even simple heating by a heating kettle (reaction kettle) using pressurized steam or a heat exchange device (plate type heat exchanger, spiral type heat exchanger, etc.) is heating using microwaves. Also good. In the heat exchange device, it is preferable that the heat exchange surface area be as large as possible in order to shorten the heating time. For example, the pH-adjusted precursor aqueous solution is introduced into the spiral conduit while controlling the flow rate, and the surroundings of the conduit are heated to a constant temperature with pressurized steam, thereby maintaining the heating time constant. Is possible. In the case of using a microwave, the irradiation conditions are not particularly limited. For example, it is preferable to irradiate a microwave of 2450 ± 30 MHz with an output of preferably 30 W or more, more preferably 100 to 400 W.
前記工程（３）における加熱温度は、１００〜１５０℃、好ましくは１２０〜１５０℃である。この温度範囲において所定時間加熱することにより、チャフロサイド類の反応収率を大きく高めることができる。これより高温で加熱すると、チャフロサイド類の熱分解が加速的に進行し始め、結果的に反応収率低減の一因となる。また、これより低い温度では十分な量のチャフロサイド類を短時間で生成させることは難しい。 The heating temperature in the said process (3) is 100-150 degreeC, Preferably it is 120-150 degreeC. By heating in this temperature range for a predetermined time, the reaction yield of chafurosides can be greatly increased. When heated at a temperature higher than this, the pyrolysis of the chafuroside begins to proceed at an accelerated rate, resulting in a reduction in the reaction yield. Moreover, it is difficult to generate a sufficient amount of chafuroside in a short time at a temperature lower than this.
チャフロサイド類の熱分解を抑え、反応収率を上げるためには、加熱時間も重要であり、反応水溶液のｐＨや加熱温度に応じて適宜決定すればよいが、好ましくは、１分〜１６分間で、これより短い時間では十分な量のチャフロサイド類の生成は認められず、これより長い時間ではチャフロサイド類の分解による反応収率の低下が懸念される。ただし、１００〜１３０℃においては比較的分解反応が起こりにくいので、必要に応じ１６分以上加熱したほうが反応収率向上に繋がることもある。 In order to suppress the thermal decomposition of the chafuroside and increase the reaction yield, the heating time is also important, and may be appropriately determined according to the pH of the aqueous reaction solution and the heating temperature, but preferably 1 minute to 16 minutes. In the shorter time, the production of a sufficient amount of chafurosides is not observed, and in the longer time, there is a concern that the reaction yield is lowered due to the decomposition of the chafurosides. However, since a decomposition reaction is relatively unlikely to occur at 100 to 130 ° C., heating for 16 minutes or longer as necessary may lead to an improvement in reaction yield.
以下に、実施例を挙げて本発明を具体的に説明するが、本発明はこれらの実施例にのみ限定されるものではない。 EXAMPLES Hereinafter, the present invention will be specifically described with reference to examples, but the present invention is not limited to these examples.
以下の実施例において用いたチャフロサイドＡとＢの前駆体（イソビテキシン２”−サルフェイトとビテキシン２”−サルフェイト）は、前記特許文献３に記載の方法で、それぞれ、イソビテキシン、ビテキシンから合成した。 The precursors of chafuroside A and B (isovitexin 2 ″ -sulfate and vitexin 2 ″ -sulfate) used in the following examples were synthesized from isovitexin and vitexin, respectively, by the method described in Patent Document 3.
本発明におけるチャフロサイド類及びその前駆体の定量は、特開２００９−１３１１６１号公報記載のＨＰＬＣ−ＭＳ／ＭＳ分析法で行い、ＨＰＬＣ−ＭＳ／ＭＳ分析には、商品名「Ａｇｉｌｅｎｔ １１００」及び「ＡＰＩ ２０００」（Ａｐｐｌｉｅｄ Ｂｉｏ社製）を併用した。ＨＰＬＣ用のカラムとしては、インタクト株式会社製のＣ１８カラムを用い、特定の溶媒を用いる「Ｃａｄｅｎｚａ ＣＤ−Ｃ１８のＨＰＬＣ−ＭＳ/ＭＳ分析法」で行った。 The quantification of chafurosides and their precursors in the present invention is performed by the HPLC-MS / MS analysis method described in JP-A-2009-131161. For HPLC-MS / MS analysis, trade names “Agilent 1100” and “API” are used. 2000 "(Applied Bio) was used in combination. As a column for HPLC, a C18 column manufactured by Intact Co., Ltd. was used, and “HPLC-MS / MS analysis method of Cadenza CD-C18” using a specific solvent was performed.
チャフロサイドＡとＢ、及びチャフロサイドＡとＢの前駆体（イソビテキシン２”−サルフェイトとビテキシン２”−サルフェイト）の検量線の作成は次のようにして行った。すなわち、予め合成したチャフロサイドＡとＢ、及びチャフロサイドＡとＢの前駆体から、それぞれ５．０ｎｇ／ｍｌ、５０ｎｇ／ｍｌ、５００ｎｇ／ｍｌ及び５０００ｎｇ／ｍｌの標準溶液を調製した。ＨＰＬＣ分析においては、これらの各標準溶液１０μｌを使用し、カラムには商品名「Ｃａｄｅｎｚａ ＣＤ−Ｃ１８」（３×１５０ｍｍ）を用い、溶出展開はＨ２Ｏ−ＣＨ３ＣＮの混合溶媒を用いて２０分かけて１５〜５０％とするグラジエント法を使用した。得られたクロマトグラムの各化合物のピーク面積より検量線を作成した。該検量線をもとに、実施例における各サンプル中の上記化合物の定量分析を行った。 Calibration curves for chafloside A and B and precursors of chafloside A and B (isovitexin 2 "-sulfate and vitexin 2" -sulfate) were prepared as follows. That is, standard solutions of 5.0 ng / ml, 50 ng / ml, 500 ng / ml and 5000 ng / ml were prepared from chafloside A and B and precursors of chafloside A and B, which were synthesized in advance. In the HPLC analysis, 10 μl of each of these standard solutions is used, the trade name “Cadenza CD-C18” (3 × 150 mm) is used for the column, and elution development is performed using a mixed solvent of H 2 O—CH 3 CN. A gradient method of 15-50% over 20 minutes was used. A calibration curve was created from the peak area of each compound in the obtained chromatogram. Based on the calibration curve, the above compounds in each sample in the examples were quantitatively analyzed.
以下の各実施例においては、特記しない限り、チャフロサイド類の前駆体を所定の濃度になるように水に溶解し、次いで、酸、塩基、塩のいずれか、若しくはそれらの混合物を加えて所定のｐＨに調整した。例えば、クエン酸−リン酸緩衝液を用いて、チャフロサイドＡ前駆体濃度が１５１０ｎｇ／ｍｌであるｐＨ６．６の試験溶液を調整する場合の典型例は次のようである：蒸留水を用いて、チャフロサイドＡ前駆体の濃度が３０２０ｎｇ／ｍｌである水溶液を作る。一方で、０．１Ｍクエン酸水溶液１３．６ｍｌと、０．２Ｍリン酸水素二ナトリウム水溶液３６．４ｍｌを混合する（合計５０ｍｌ）。この混合液に、チャフロサイドＡ前駆体濃度が３０２０ｎｇ／ｍｌである前記の水溶液５０ｍｌを加えて混合することにより、チャフロサイドＡ前駆体濃度が１５１０ｎｇ／ｍｌであるｐＨ６．６の試験溶液を得ることが出来る。通常、ｐＨは６．６から外れることはないが、もしも少し外れた値になったら、０．１Ｍクエン酸水溶液または０．２Ｍリン酸水素二ナトリウム水溶液を少量ずつ添加し、ｐＨを６．６に合わせる。 In each of the following examples, unless otherwise specified, a chafuroside precursor is dissolved in water to a predetermined concentration, and then an acid, a base, a salt, or a mixture thereof is added to the predetermined concentration. Adjusted to pH. For example, a typical example of preparing a pH 6.6 test solution having a chafuroside A precursor concentration of 1510 ng / ml using a citrate-phosphate buffer is as follows: An aqueous solution having a chafloside A precursor concentration of 3020 ng / ml is prepared. On the other hand, 13.6 ml of 0.1M aqueous citric acid solution and 36.4 ml of 0.2M aqueous solution of disodium hydrogenphosphate are mixed (total of 50 ml). By adding 50 ml of the aqueous solution having a chafuroside A precursor concentration of 3020 ng / ml to this mixed solution and mixing, a test solution having a pH of 6.6 having a chafuroside A precursor concentration of 1510 ng / ml can be obtained. . Normally, the pH does not deviate from 6.6, but if it becomes slightly deviated, 0.1M aqueous citric acid solution or 0.2M aqueous disodium hydrogenphosphate solution is added little by little to adjust the pH to 6.6. To match.
チャフロサイド類の前駆体（イソビテキシン２”−サルフェイト、ビテキシン２”−サルフェイト）の濃縮混合物は次のようにして調製した。茶葉（１ｋｇ、品種：鳳凰水仙）を蒸留水（５０００ｍｌ）に浸漬し、８０℃で３０分間攪拌した。不溶物を濾去した後、水抽出液体積の約半量のｎ−ＢｕＯＨを加え、液液分配を行った。この操作を２回行い、得られた水画分を、希塩酸を用いてｐＨ４．０とした後にＳｅｐａｂｅａｓ
ＳＰ８２５(ｍｉｔｓｕｂｉｓｈｉ ｃｈｅｍｉｃａｌ)カラムクロマトグラフィーに付し、水、２０％ＭｅＯＨ、５０％ＭｅＯＨ、１００％ＭｅＯＨで順次溶出した。次いで、チャフロサイド類の前駆体を含む５０％ＭｅＯＨ溶出部（１６０ｇ）をＣＨＣｌ３−ＭｅＯＨ−Ｈ２Ｏ(６０：４０：８)を展開溶媒とするＳｉＯ２カラムクロマトグラフィーに付し、チャフロサイドＡ及びＢの前駆体の濃縮混合物（２２ｇ）を得た。本濃縮混合物中の両前駆体の合計含有量は約１０％で、その割合は、チャフロサイドＡ前駆体：Ｂ前駆体＝１：１．１であった。さらに、この濃縮混合物をＯＤＳカラムクロマトグラフィーに付して精製することにより、チャフロサイドＡ前駆体０．９６ｇとチャフロサイドＢ前駆体１．０４ｇを得た。
A concentrated mixture of chafuroside precursors (isovitexin 2 ″ -sulfate, vitexin 2 ″ -sulfate) was prepared as follows. Tea leaves (1 kg, cultivar: Shosuisen) were immersed in distilled water (5000 ml) and stirred at 80 ° C. for 30 minutes. After insoluble matter was removed by filtration, about half of the volume of the water extract was added to n-BuOH to perform liquid-liquid partition. This operation was performed twice, and the obtained water fraction was adjusted to pH 4.0 with dilute hydrochloric acid and then separated into Sephaeas.
The column was subjected to SP825 (mitsubishi chemical) column chromatography and eluted sequentially with water, 20% MeOH, 50% MeOH and 100% MeOH. Next, a 50% MeOH elution part (160 g) containing a precursor of chafurosides was subjected to SiO 2 column chromatography using CHCl 3 —MeOH—H 2 O (60: 40: 8) as a developing solvent. A concentrated mixture (22 g) of the precursor of B was obtained. The total content of both precursors in the concentrated mixture was about 10%, and the ratio was chafuroside A precursor: B precursor = 1: 1.1. Further, the concentrated mixture was purified by ODS column chromatography to obtain 0.96 g of chafuroside A precursor and 1.04 g of chafuroside B precursor.
本参考例において用いた茶抽出物は次のようにして調製した。すなわち、市販の各茶葉を粉砕器（イワタニミルサー、岩谷産業株式会社製）で粉砕し、各２５ｇに蒸留水１０００ｍｌを加え、８０℃で３０分間抽出した。その茶抽出液に含まれるチャフロサイドＡの前駆体（イソビテキシン２”−サルフェイト）とチャフロサイドＡの含有量を定量した。これらの抽出液を必要に応じて蒸留水で希釈し、所定の濃度、所定のｐＨに調整して用いた。 The tea extract used in this reference example was prepared as follows. That is, each commercially available tea leaf was pulverized with a pulverizer (Iwatani Milcer, manufactured by Iwatani Corporation), 1000 ml of distilled water was added to each 25 g, and extracted at 80 ° C. for 30 minutes. The contents of the precursor of chafuroside A (isovitexin 2 ″ -sulfate) and chafuroside A contained in the tea extract were quantified. These extracts were diluted with distilled water as necessary to obtain a predetermined concentration and a predetermined concentration. The pH was adjusted to be used.
本参考例で用いた茶エキスｎｅａｔ物質は次のようにして調製した。すなわち、茶葉をミルサー（岩谷産業株式会社製）で粉砕後、粉砕物１ｇ／溶媒１００ｍｌの割合になるように水または５０％メタノールを加え、８０℃で３０分間、攪拌下抽出した。次いで、上記チャフロサイドＡ及びＢの前駆体の濃縮混合物を添加し、チャフロサイドＡ及びＢの前駆体の高含有茶水溶液、及びチャフロサイドＡ及びＢの前駆体高含有５０％メタノールを作成した。チャフロサイドＡ及びＢの前駆体高含有５０％メタノール溶液を濃縮乾固し、エキスｎｅａｔ物質として用いた。 The tea extract neat substance used in this reference example was prepared as follows. That is, tea leaves were pulverized with a miller (manufactured by Iwatani Sangyo Co., Ltd.), water or 50% methanol was added at a ratio of 1 g of pulverized product / 100 ml of solvent, and extraction was performed with stirring at 80 ° C for 30 minutes. Subsequently, the concentrated mixture of the precursors of the chaflosides A and B was added to prepare a high content tea aqueous solution of the precursors of the chaflosides A and B and 50% methanol containing the precursors of the chaflosides A and B. A 50% methanol solution containing a high amount of precursors of chafuroside A and B was concentrated to dryness and used as an extract neat substance.
本実施例における加熱処理には、加熱装置付き油浴（Ｎｉｓｓｉｎ社製ＮＷＢ−１２０Ｎ）、マイクロウェーブ加熱装置（Ｂｉｏｔａｇｅ社製Ｉｎｉｔｉａｔｏｒ８）、あるいはプレート型熱交換器（日阪製作所製）を使用した。
実施例１
For the heat treatment in this example, an oil bath with a heating device (NWB-120N manufactured by Nissin), a microwave heating device (Initiator 8 manufactured by Biotage), or a plate heat exchanger (manufactured by Nisaka Seisakusho) was used.
Example 1
チャフロサイドＡ前駆体（イソビテキシン２”−サルフェイト）を所定量溶かした水溶液に炭酸ナトリウム水溶液を加え、前駆体濃度が１０４０ｎｇ／ｍｌ、ｐＨが１０．６となるように調整した。本ｐＨ調整済水溶液を、マイクロウェーブ加熱装置を用いて、１００℃〜１５０℃の範囲（１０℃間隔）で２分間加熱した（実施例１）。比較例１として、チャフロサイドＡ前駆体を水溶液とすることなくｎｅａｔ状のまま、油浴中、１００℃〜１５０℃の範囲（１０℃間隔）で４分間加熱した。また、比較例２として、チャフロサイドＡ前駆体の水溶液をｐＨ調整することなく、油浴中、１３０℃〜１５０℃の範囲（１０℃間隔）で４分間加熱した。参考例１として、茶（品種：蜜蘭香）エキスｎｅａｔ物質をｎｅａｔ状のまま、油浴中、１３０℃で２分〜１６分間加熱した。また、参考例２として、茶（品種：水仙）エキスｎｅａｔ物質をｎｅａｔ状のまま、油浴中で４分間、参考例３として、茶（品種：水仙）抽出物の水溶液をｐＨ未調整のまま（未調整でｐＨ５．２）、マイクロウェーブ加熱で２分間、それぞれ１００℃〜１５０℃の範囲（１０℃間隔）で加熱した。得られた各サンプルをＨＰＬＣ−ＭＳ／ＭＳ分析に付し、原料のイソビテキシン２”−サルフェイトと生成物であるチャフロサイドＡの含有量を定量した。結果を表１に記す。 A sodium carbonate aqueous solution was added to an aqueous solution in which a predetermined amount of chafuroside A precursor (isovitexin 2 ″ -sulfate) was dissolved, and the precursor concentration was adjusted to 1040 ng / ml and the pH was 10.6. Was heated for 2 minutes in the range of 100 ° C. to 150 ° C. (interval of 10 ° C.) using a microwave heating apparatus (Example 1) As Comparative Example 1, the neat shape without using the chafuroside A precursor as an aqueous solution In the oil bath, it was heated for 4 minutes in the range of 100 ° C. to 150 ° C. (at intervals of 10 ° C.) As Comparative Example 2, the aqueous solution of the Chafloside A precursor was adjusted to 130 in the oil bath without adjusting the pH. The sample was heated for 4 minutes in the range of 10 ° C. to 150 ° C. As Reference Example 1, the tea (variety: honey orchid) extract neat substance was kept neat in an oil bath, 1 The mixture was heated at 0 ° C. for 2 to 16 minutes, and as Reference Example 2, tea (variety: narcissus) extract neat substance was kept neat for 4 minutes in oil bath, and as Reference Example 3, tea (variety: narcissus). ) The aqueous solution of the extract was heated in the range of 100 ° C. to 150 ° C. (10 ° C. intervals) for 2 minutes with microwave heating while the pH was not adjusted (pH 5.2 without adjustment). By subjecting to HPLC-MS / MS analysis, the content of the raw material isovitexin 2 ″ -sulfate and the product chafuroside A was quantified. The results are shown in Table 1.
なお、表中記載の「前Ａ」、「Ａ」は、それぞれイソビテキシン２”−サルフェイト、チャフロサイドＡを表し、「ＭＷ」は、マイクロウェーブ加熱であることを示す。「−」は未実施を示す。また、表中に示される各化合物の定量値の単位は、ｎｇ／ｍｌである。以下同様。 In the table, “Previous A” and “A” represent isovitexin 2 ″ -sulfate and chafuroside A, respectively, and “MW” represents microwave heating. "-" Indicates not implemented. The unit of the quantitative value of each compound shown in the table is ng / ml. The same applies hereinafter.
表１においては、外部から何も加えることなく、ｎｅａｔ状のまま、あるいは水溶液で加熱した場合の反応性と、外部から塩を添加した水溶液で反応した場合の比較結果を示す。茶エキスｎｅａｔ物質をｎｅａｔ状のまま、油浴中、１３０℃で加熱した参考例１においては、チャフロサイドＡの定量値は若干増えているものの、原料である前駆体の量が事実上減少していないことから、チャフロサイドＡの数値変化は、ｎｅａｔ物質のどの部分を定量に用いたかによる測定誤差の範囲であると思われる。すなわち、何も添加しないで加熱した場合には、少なくとも加熱時間１６分の範囲ではチャフロサイドＡが実質生成しないことが明らかとなった。次に、品種は異なるが同じく茶エキスｎｅａｔ物質をｎｅａｔ状のまま、油浴中で４分間、今度は加熱温度を１００℃〜１５０℃の範囲で変化させ、その影響を調べた（参考例２）。１３０℃〜１４０℃では、前駆体の減少量に対応する分だけ僅かにチャフロサイドＡが増加しており、このくらいの温度から反応が進行することが判る。１５０℃における反応収率（チャフロサイドＡ生成モル数／前駆体初期モル数）は約６．８％であった（この場合の収率を具体的計算例として示すと、チャフロサイドＡ前駆体の分子量は５１２、チャフロサイドＡの分子量は４１４なので、{（９５−５９）／４１４}／（６５０／５１２）＝０．０８７／１．２７０＝６．８％となる。以下同様）。この結果から、ｎｅａｔ物質をｎｅａｔ状のまま無添加で反応しても、反応温度が１５０℃以上になれば反応が進行することが判る。一方、水溶液にした場合の反応性を調べたのが参考例３である。水溶液での反応も、基本的にはｎｅａｔ状での反応結果と同じで、１５０℃以上で反応が進行するようになり、１５０℃の収率は４．６％であった。次に、茶抽出物ではなく、前駆体イソビテキシン２”−サルフェイトを用い、無添加、ｎｅａｔ状態で、油浴中４分間の加熱を行った（比較例１）。結果は上記参考例２とほぼ同じで、１５０℃で収率１２％を示した。また、前駆体の水溶液をｐＨ未調整のまま加熱した場合（比較例２）の結果も、比較例１と同じパターンを示した（１５０℃で収率１１％）。以上から、ｎｅａｔ状であれ水溶液状であれ、外部からの添加物なしの条件では、１５０℃未満の温度では事実上反応は進行しない、若しくは極めて進行し難く、２〜４分間の加熱では１０数％以下の収率しか得られなかった。 Table 1 shows the comparison results when reacting with an aqueous solution to which salt is added from the outside, with the reactivity when heated in an aqueous solution without adding anything from the outside. In Reference Example 1 in which the tea extract neat substance was heated in an oil bath at 130 ° C. in the neat form, the quantitative value of chafuroside A was slightly increased, but the amount of precursor as a raw material was substantially decreased. Therefore, it is considered that the numerical change of chafloside A is within the range of measurement error depending on which part of the neat substance is used for quantification. That is, when heating was carried out without adding anything, it became clear that chafloside A was not substantially formed in the range of at least a heating time of 16 minutes. Next, although the varieties were different, the tea extract neat substance was kept in the neat state, and the heating temperature was changed in the range of 100 ° C. to 150 ° C. for 4 minutes in the oil bath, and the influence was examined (Reference Example 2). ). At 130 ° C. to 140 ° C., chafloside A slightly increases by the amount corresponding to the amount of decrease in the precursor, and it can be seen that the reaction proceeds from this temperature. The reaction yield at 150 ° C. (number of moles of chafuroside A formed / number of moles of initial precursor) was about 6.8% (when the yield in this case is shown as a specific calculation example, the molecular weight of chafuroside A precursor is 512, and the molecular weight of chafuroside A is 414, so {(95-59) / 414} / (650/512) = 0.087 / 1.270 = 6.8%, and so on. From this result, it can be seen that even if the neat substance is reacted in the neat state without addition, the reaction proceeds when the reaction temperature reaches 150 ° C. or higher. On the other hand, Reference Example 3 examined the reactivity when an aqueous solution was used. The reaction in the aqueous solution was basically the same as the reaction result in the neat state, and the reaction proceeded at 150 ° C. or higher, and the yield at 150 ° C. was 4.6%. Next, the precursor isovitexin 2 ″ -sulfate was used instead of the tea extract, and heating was performed in an oil bath for 4 minutes without addition and in a neat state (Comparative Example 1). The results were almost the same and showed a yield of 12% at 150 ° C. The result of heating the aqueous precursor solution without adjusting the pH (Comparative Example 2) also showed the same pattern as Comparative Example 1 (150 The yield is 11% at 0 ° C. From the above, the reaction does not proceed or is extremely difficult to proceed at temperatures below 150 ° C. under the condition of no neat or aqueous solution, with no external additives. Only a yield of 10% or less was obtained by heating for -4 minutes.
これに対し、チャフロサイドＡ前駆体（イソビテキシン２”−サルフェイト）の水溶液に炭酸ナトリウム水溶液を加え、ｐＨ１０．６に調整し、マイクロウェーブを用いて２分間加熱した場合、実施例１の結果から明らかなように、反応は１００℃、２分間でも僅かに進行し（収率５．１％）、１４０℃で最高収率７３％という結果を得ることが出来た。ただし、ｐＨ１０．６という強塩基性条件では、原料、生成物ともに分解が起こりやすく、１４０℃でも２０〜３０％の分解が起こることも判明した（分解率は、どちらの化合物がどれだけ分解したか不明なため正確な計算はできないので、目安として、重量基準での分解率を示した。以下同様）。 On the other hand, when the aqueous solution of chafloside A precursor (isovitexin 2 ″ -sulfate) was added with an aqueous sodium carbonate solution, adjusted to pH 10.6, and heated for 2 minutes using microwaves, it was clear from the results of Example 1. Thus, the reaction proceeded slightly even at 100 ° C. for 2 minutes (a yield of 5.1%), and a maximum yield of 73% was obtained at 140 ° C. However, a strong base of pH 10.6 was obtained. It was also found that the raw material and the product are prone to decomposition under the sexual conditions, and that 20-30% decomposition occurs even at 140 ° C. (The decomposition rate is unknown because it is unclear how much of which compound has decomposed. Since it is not possible, the decomposition rate on the basis of weight is shown as a guide.
以上、表１の結果から、製造過程における操作性を考慮して水溶液状態での反応を試みたが、ｎｅａｔ状であれ水溶液状であれ、外部からの添加物なしの条件では、１５０℃未満の温度では事実上反応は進行しない、若しくは極めて進行し難かった。しかしながら、実施例１では炭酸ナトリウムを用いたが、反応水溶液に酸、塩基、塩を添加するこることにより反応速度が大幅に促進され、１００℃〜１５０℃、とりわけ、１３０℃〜１５０℃では十分な反応収率が得られ、分解を考えなければ、加熱温度が高いほど反応速度が大きくなることを見出した。
実施例２〜７
As described above, from the results of Table 1, the reaction in the aqueous solution state was attempted in consideration of the operability in the production process. However, it was less than 150 ° C. under the condition of no neat or aqueous solution and no external additive. At temperature, the reaction did not proceed substantially or was very difficult to proceed. However, although sodium carbonate was used in Example 1, the reaction rate was greatly accelerated by adding acid, base, and salt to the aqueous reaction solution, and 100 ° C to 150 ° C, especially 130 ° C to 150 ° C was sufficient. It was found that the reaction rate was increased as the heating temperature was increased without considering the decomposition.
Examples 2-7
チャフロサイドＡ前駆体（イソビテキシン２”−サルフェイト）を所定量溶かした水溶液に酢酸緩衝液、あるいはクエン酸−リン酸緩衝液を加えてそれぞれのｐＨを５．８（実施例２〜４）、あるいは７．０（実施礼５〜７）に調整した。これらｐＨ調整済水溶液を、マイクロウェーブ加熱装置を用いて、１１０℃〜１３０℃の範囲（１０℃間隔）で１〜１６分間加熱した。得られた各サンプルを、ＨＰＬＣ−ＭＳ／ＭＳ分析に付し、原料のイソビテキシン２”−サルフェイトと生成物であるチャフロサイドＡの含有量を定量した。結果を表２に記す。 Acetic acid buffer solution or citrate-phosphate buffer solution is added to an aqueous solution in which a predetermined amount of chafuroside A precursor (isovitexin 2 ″ -sulfate) is dissolved, and each pH is 5.8 (Examples 2 to 4), or The pH adjusted aqueous solution was heated in the range of 110 ° C. to 130 ° C. (10 ° C. interval) for 1 to 16 minutes using a microwave heating apparatus. Each of the obtained samples was subjected to HPLC-MS / MS analysis, and the content of the raw material isovitexin 2 ″ -sulfate and the product chafuroside A was quantified. The results are shown in Table 2.
実施例２〜４及び実施例５〜７においては、外部からの酸、塩基、あるいは塩が、実施例１のような塩でない場合でも、チャフロサイド前駆体からチャフロサイドへの変換が上手く進行するか否かを調べた。すなわち、添加物の種類とｐＨの影響について比較検討した。表２から１１０〜１３０℃、ｐＨ５．８〜７．０の条件では、反応温度が高いほど、そして反応時間が長いほど反応収率が向上すること（１３０℃、１６分では若干の分解が起こっているが）、そしてｐＨが高いほど反応速度が大きいことが一目瞭然である。具体的には、実施例２〜４におけるｐＨ５．８、加熱時間１６分の結果を比較するに、反応収率は１１０℃では２４％、１２０℃では５１％、１３０℃では８０％と反応温度依存的に収率が上がっている。また、この１３０℃、１６分では分解も少なく僅か７％弱に過ぎない。この結果から推定するに、もしも更に反応時間を１６分延長すれば、計算上は、{（４２７−２）＋１８３×０．８０}}×０．９３＝５３１ｎｇ／ｍｌのチャフロサイドＡが生成することになる。この場合の推定収率は、（５３１／４１４）／（６５１／５１２）＝１００％という数字になる。同様にしてｐＨ７．０における実施例５〜７では、ｐＨ５．８に比べ、反応速度が大きく、分解が起こり難い条件下では３〜４倍速い速度で反応が進行している。そして、加熱時間１６分の反応収率は、１１０℃では６８％、１２０℃では８８％、そして１３０℃では分解のため収率が落ち７７％（加熱８分で収率９０％を達成）となっている。１２０℃、１６分、あるいは、１３０℃、８分ではいくらかの分解が起こっていることを考えれば極めて高い収率であると言える。 In Examples 2 to 4 and Examples 5 to 7, even if the external acid, base, or salt is not the salt as in Example 1, the conversion from the chafuroside precursor to chafuroside proceeds well. I investigated. That is, the type of additive and the effect of pH were compared. From Table 2, under the conditions of 110 to 130 ° C. and pH 5.8 to 7.0, the higher the reaction temperature and the longer the reaction time, the better the reaction yield (slight decomposition occurs at 130 ° C. for 16 minutes). It is obvious that the higher the pH, the greater the reaction rate. Specifically, comparing the results of pH 5.8 and heating time of 16 minutes in Examples 2 to 4, the reaction yield was 24% at 110 ° C, 51% at 120 ° C, and 80% at 130 ° C. The yield is dependently increased. In addition, at 130 ° C. for 16 minutes, there is little decomposition and only 7%. From this result, if the reaction time is further extended by 16 minutes, {(427-2) + 183 × 0.80}} × 0.93 = 531 ng / ml of chafuroside A is generated in the calculation. become. The estimated yield in this case is the number (531/414) / (651/512) = 100%. Similarly, in Examples 5 to 7 at pH 7.0, the reaction proceeds at a rate 3 to 4 times faster under the conditions where the reaction rate is larger and decomposition is difficult to occur than at pH 5.8. The reaction yield of 16 minutes at heating time is 68% at 110 ° C, 88% at 120 ° C, and 77% at 130 ° C due to decomposition, resulting in a yield of 77% (achieving 90% in 8 minutes of heating). It has become. Considering that some decomposition occurs at 120 ° C. for 16 minutes or 130 ° C. for 8 minutes, it can be said that the yield is extremely high.
以上、表２の結果から、外部から添加する物質は、実施例１における炭酸ナトリウムのような塩である必要は無く、酸と塩の組み合わせでもよく、その酸と塩は通常使用する酸、塩であれば種類には無関係であり、また、水溶液の液性は塩基性である必要は無く、酸性でも中性でもよく、ｐＨ値が高いほど反応速度が大きいこと、そして、加熱時間１６分以内に収率８０％以上を達成できることが明らかになった。
実施例８〜１７
As described above, from the results of Table 2, the substance added from the outside need not be a salt such as sodium carbonate in Example 1, and may be a combination of an acid and a salt. The acid and the salt are usually used acids and salts. As long as the aqueous solution does not need to be basic, it may be acidic or neutral, and the higher the pH value, the higher the reaction rate, and the heating time is within 16 minutes. It was revealed that a yield of 80% or more can be achieved.
Examples 8-17
チャフロサイドＡ前駆体（イソビテキシン２”−サルフェイト）を所定量溶かした水溶液に各種緩衝液、塩又は塩基を加え、それぞれのｐＨを２．４〜１１．７に調整した。これらｐＨ調整済水溶液を、マイクロウェーブ加熱装置を用いて、１００℃〜１９０℃、若しくは８０℃〜１８０℃（又は１７０℃）の範囲（１０℃間隔）で２分間加熱した。得られた各サンプルを、ＨＰＬＣ−ＭＳ／ＭＳ分析に付し、原料のイソビテキシン２”−サルフェイトと生成物であるチャフロサイドＡの含有量を定量した。結果を表３に記す。 Various buffers, salts, or bases were added to an aqueous solution in which a predetermined amount of chafuroside A precursor (isovitexin 2 ″ -sulfate) was dissolved, and the pH of each was adjusted to 2.4 to 11.7. The sample was heated for 2 minutes in the range of 100 ° C. to 190 ° C., or 80 ° C. to 180 ° C. (or 170 ° C.) at intervals of 10 ° C. Each sample obtained was subjected to HPLC-MS / By subjecting to MS analysis, the contents of the raw material isovitexin 2 ″ -sulfate and the product chafuroside A were quantified. The results are shown in Table 3.
実施例８〜１７（及び実施例２）においては、これまでの結果を踏まえ、マイクロウェーブを用いた２分間の加熱条件における好ましいｐＨの設定範囲と加熱時間との関係について検証した。すなわち、反応に付す水溶液のｐＨを、ｐＨ２．４の強酸性条件から、ｐＨ１１．７の強塩基性条件まで振り、主として各ｐＨにおける１００〜１５０℃における反応収率を確認した。以下、各実施例の結果について触れる。実施例８及び９の結果から、ｐＨ５未満においては事実上チャフロサイドＡが生成しないこと、また、ｐＨ２．４では反応温度の上昇に伴い原料が減少、とりわけ１６０℃以上では急激に減少するが、これは原料の脱硫酸化が起こり、対応する脱硫酸化体（イソビテキシン）が生じていることを確認した（データ未開示）。一方、ｐＨ４．０以上においてはこのような脱硫酸化が事実上起こっていないことも確認した。実施例９のｐＨ４．０においても１６０℃以上では脱硫酸化とは異なる分解が起こるものの、温度が上がるに従って収率は向上し、１９０℃では収率４２％になった。さらにｐＨを上げ、ｐＨ５．０以上になると反応性は大きく向上してくる。具体的には、ｐＨ５．０（実施例１０）、５．８（実施例１１）、６．６（実施例１２）の酸性側においては、ｐＨが上がるにしたがって最高収率が得られる反応温度は、１７０℃、１６０℃、１５０℃と低くなり、反応収率もそれに合わせて、７２％、８２％、８８％と向上した。反応温度１５０℃での収率も当然ｐＨに合わせて上昇し、３２％、７７％、８８％であった。実施例１３〜１６のグリシン／水酸化ナトリウムを用いたｐＨ７．５〜１０．０の系においては少々様相が変化し、ｐＨ７．５、ｐＨ８．３における１５０℃の反応収率は、それぞれ１０％、４１％と大きく低下した。このような結果となった理由は定かではないが、両性物質であるグリシンを用いたことに一因があるようにも思われる。ちなみに、茶エキスを原料として用いた場合の結果ではあるが、リン酸二水素ナトリウム／リン酸水素二ナトリウムを用いたｐＨ７．８、１４０℃、２分での収率は７３％、あるいは炭酸ナトリウム／重炭酸ナトリウムを用いたｐＨ８．６、１３０℃、２分では収率６７％であった。グリシン／水酸化ナトリウムを用いてｐＨ調整した場合でも、更に高いｐＨ領域においては十分な収率が得られた。すなわち、加熱温度１５０℃における収率は、ｐＨ９．０では７９％（実施例１５）、ｐＨ１０．０では９２％（実施例１６）であった。さらにｐＨが高い場合として、炭酸ナトリウムを添加したｐＨ１０．６の水溶液（実施例２）、水酸化ナトリウムを添加したｐＨ１１．７の水溶液（実施例１７）での反応性と収率を見るに、１００℃以下の温度における目的物の増加量から、これらの強塩基性ｐＨ条件では反応速度は更に促進されていると思われる。そして、１５０℃以下の温度においても分解が顕著であるにも拘らず、ｐＨ１０．６、１４０℃では、反応収率７３％という数字が出ている。また、ｐＨ１１．７では、さらに低い温度の１００℃で収率１５％、１２０℃で収率４６％であった。 In Examples 8 to 17 (and Example 2), based on the results so far, the relationship between the preferable pH setting range and the heating time in a heating condition for 2 minutes using a microwave was verified. That is, the pH of the aqueous solution subjected to the reaction was shaken from a strongly acidic condition of pH 2.4 to a strongly basic condition of pH 11.7, and the reaction yield at 100 to 150 ° C. at each pH was mainly confirmed. Hereinafter, the results of each example will be described. From the results of Examples 8 and 9, it can be seen that virtually no chafuroside A is formed at a pH lower than 5, and the raw material decreases with an increase in the reaction temperature at pH 2.4, particularly rapidly at 160 ° C. or higher. Confirmed that the raw material was desulfated and the corresponding desulfated form (isovitexin) was formed (data not disclosed). On the other hand, it was also confirmed that such desulfation did not occur at pH 4.0 or higher. Even at pH 4.0 of Example 9 at 160 ° C. or higher, decomposition different from that of desulfation occurred, but the yield increased as the temperature increased, and at 190 ° C., the yield was 42%. When the pH is further raised to pH 5.0 or higher, the reactivity is greatly improved. Specifically, on the acidic side of pH 5.0 (Example 10), 5.8 (Example 11), and 6.6 (Example 12), the reaction temperature at which the highest yield is obtained as the pH increases. Decreased to 170 ° C., 160 ° C., and 150 ° C., and the reaction yields were improved to 72%, 82%, and 88% accordingly. Naturally, the yield at a reaction temperature of 150 ° C. also increased with the pH, and was 32%, 77%, and 88%. In the system of pH 7.5 to 10.0 using glycine / sodium hydroxide of Examples 13 to 16, the appearance was slightly changed, and the reaction yield at 150 ° C. at pH 7.5 and pH 8.3 was 10% respectively. , 41%. The reason for this result is not clear, but it seems to be due to the use of amphoteric glycine. By the way, although it is a result when using tea extract as a raw material, pH 7.8 using sodium dihydrogen phosphate / disodium hydrogen phosphate at 140 ° C. for 2 minutes is 73%, or sodium carbonate / PH 8.6 using sodium bicarbonate, 130 ° C., 2 minutes, yield was 67%. Even when the pH was adjusted with glycine / sodium hydroxide, a sufficient yield was obtained in a higher pH range. That is, the yield at a heating temperature of 150 ° C. was 79% (Example 15) at pH 9.0 and 92% (Example 16) at pH 10.0. In order to see the reactivity and yield in a pH 10.6 aqueous solution (Example 2) to which sodium carbonate was added and a pH 11.7 aqueous solution (Example 17) to which sodium hydroxide was added as a case where the pH was higher, From the increased amount of the target product at a temperature of 100 ° C. or lower, it seems that the reaction rate is further accelerated under these strongly basic pH conditions. And although decomposition | disassembly is remarkable also at the temperature of 150 degrees C or less, the number of reaction yield 73% has come out at pH 10.6 and 140 degreeC. At pH 11.7, the yield was 15% at a lower temperature of 100 ° C, and the yield was 46% at 120 ° C.
表３の結果は総じてこれまでの結果を追認するものであるが、反応温度が上がると反応速度も上昇するが、一方では分解反応も起こり易くなり、結果的に反応収率を下げる結果となる場合もあることを示している。とりわけ、ｐＨが１０を越えると、１５０℃以下においても分解が顕著になり、反応収率の低下に繋がることが明らかとなった。
実施例１８
The results in Table 3 generally confirm the results so far, but as the reaction temperature increases, the reaction rate also increases, but on the other hand, the decomposition reaction tends to occur, resulting in a decrease in the reaction yield. It shows that it may be. In particular, when the pH exceeded 10, it became clear that the decomposition became remarkable even at 150 ° C. or lower, leading to a decrease in the reaction yield.
Example 18
チャフロサイドＡ前駆体（イソビテキシン２”−サルフェイト）を所定量溶かした水溶液（１ｍｌ）に、水酸化ナトリウム水溶液（濃度２０ｍｇ／ｍｌ）を各々２５μｌ、５０μｌ、ｌ００μｌ加えた。この際の各水溶液のｐＨは、ｐＨメーターの測定範囲（ｐＨ１２）を超えていて測定できないため、不明である。これらの水溶液を、マイクロウェーブ加熱装置を用いて、１１０℃で２分間加熱した。得られた各サンプルを、ＨＰＬＣ−ＭＳ／ＭＳ分析に付し、原料のイソビテキシン２”−サルフェイトと生成物であるチャフロサイドＡの含有量を定量した。結果を表４に記す。 25 μl, 50 μl and 100 μl of an aqueous sodium hydroxide solution (concentration 20 mg / ml) were added to an aqueous solution (1 ml) in which a predetermined amount of chafuroside A precursor (isovitexin 2 ″ -sulfate) was dissolved. The pH of each aqueous solution at this time Is unknown because it exceeds the measurement range of the pH meter (pH 12), and these aqueous solutions were heated for 2 minutes at 110 ° C. using a microwave heating apparatus. By subjecting to HPLC-MS / MS analysis, the content of the raw material isovitexin 2 ″ -sulfate and the product chafuroside A was quantified. The results are shown in Table 4.
実施例１８においては、反応水溶液のｐＨ値の上限を確認するために実施したものである。結果的には、水酸化ナトリウム水溶液（濃度２０ｍｇ／ｍｌ）の２５μｌ添加においても、重量基準で約５０％の分解が起こっており、反応収率は３７％であった。それ以上濃い水酸化ナトリウムの存在下では、更に分解速度が速く、反応収率はさらに低いものとなった（５０μｌ添加では収率２８％、１００μｌ添加では収率４％）。反応溶液の取扱の容易さや、グラスライニングの反応釜を使用し難いこと、あるいは中和に大量の酸を要すること等を考慮するに、反応条件としては必ずしも好ましいものではないといえる。
実施例１９
In Example 18, it implemented in order to confirm the upper limit of pH value of reaction aqueous solution. As a result, even when 25 μl of an aqueous sodium hydroxide solution (concentration 20 mg / ml) was added, decomposition of about 50% occurred on a weight basis, and the reaction yield was 37%. In the presence of more concentrated sodium hydroxide, the decomposition rate was faster and the reaction yield was even lower (28% yield with 50 μl addition, 4% yield with 100 μl addition). Considering the ease of handling the reaction solution, the difficulty of using a glass-lined reaction kettle, or the need for a large amount of acid for neutralization, it can be said that the reaction conditions are not necessarily preferable.
Example 19
鳳凰水仙由来のチャフロサイドＡ及びＢの前駆体の濃縮混合物（混合比１：１．１）を用い、水溶液中の両前駆体の濃度が２倍公比となるように調整した。添加物としてクエン酸／リン酸水素二ナトリウムを用い、各ｐＨを７．０とし、マイクロウェーブで、１３０℃、２分間加熱した。得られた各サンプルを、ＨＰＬＣ−ＭＳ／ＭＳ分析に付し、各成分を定量した。結果を表５に記す。 Using a concentrated mixture (mixing ratio 1: 1.1) of the precursors of Chafloside A and B derived from Daffodil, the concentration of both precursors in the aqueous solution was adjusted to a double common ratio. Citric acid / disodium hydrogen phosphate was used as an additive, each pH was set to 7.0, and it was heated in a microwave at 130 ° C. for 2 minutes. Each sample obtained was subjected to HPLC-MS / MS analysis to quantify each component. The results are shown in Table 5.
なお、表中記載の「前Ａ」、「前Ｂ」、「Ａ」、「Ｂ」は、それぞれイソビテキシン２”−サルフェイト、ビテキシン２”−サルフェイト、チャフロサイドＡ、チャフロサイドＢを表す。 In the table, “front A”, “front B”, “A”, and “B” represent isovitexin 2 ″ -sulfate, vitexin 2 ″ -sulfate, chafloside A, and chafloside B, respectively.
実施例１９においては、チャフロサイドＡ及びＢ前駆体の濃縮混合物を用い、希薄溶液から高濃度溶液まで反応液中の原料濃度をふり、前駆体濃度が変換効率に与える影響について検討した。併せて、チャフロサイドＢ前駆体においても、チャフロサイドＡ前駆体の場合と同様に反応が進行するか否かを検討した。また、本検討を行うことによって、反応液中に不純物として存在する供雑物（この場合は各種の抽出成分）の、反応効率に与える影響についても同時に確認した。検討した両前駆体の合計最高濃度は４０．３２μｇ／ｍｌで、前記実施例の約２５〜６０倍の濃度に相当する。表５から明らかなように、２倍公比で濃度を高めて行っても、それに比例してチャフロサイドＡ及びＢの濃度が上昇し、変換効率が減じることは全く無いことが明らかになった。すなわち、数１０μｇ／ｍｌ程度の濃度であれば全く変換効率に影響せず、さらには数１０００μｇ／ｍｌでも大きな問題は生じないであろうことが推測される。また、チャフロサイドＢの前駆体（ビテキシン２”−サルフェイト）からは２５％の収率でチャフロサイドＢが得られることが確認できた。この条件では化合物の分解は事実上起こっていないことから、さらに反応時間を延ばせばチャフロサイドＢの収率が上がることが見込まれる。更には、反応溶液中に各種の不純物が共存しても、反応効率に事実上大きな影響を与えないことが判った。
実施例２０
In Example 19, a concentrated mixture of chafuroside A and B precursors was used, the raw material concentration in the reaction solution was varied from a dilute solution to a high concentration solution, and the influence of the precursor concentration on the conversion efficiency was examined. In addition, it was examined whether the reaction proceeds in the case of the chafloside B precursor as in the case of the chafloside A precursor. In addition, by conducting this study, the influence of impurities (in this case, various extracted components) present as impurities in the reaction solution on the reaction efficiency was also confirmed. The combined maximum concentration of both precursors studied was 40.32 μg / ml, corresponding to a concentration of about 25-60 times that of the previous example. As is apparent from Table 5, even when the concentration was increased at a double common ratio, the concentrations of chafuroside A and B increased in proportion thereto, and the conversion efficiency was never decreased. That is, it is presumed that if the concentration is about several tens of μg / ml, the conversion efficiency is not affected at all, and even if it is several thousand μg / ml, no major problem will occur. Further, it was confirmed that chafuroside B was obtained in a yield of 25% from the precursor of chafuroside B (vitexin 2 ″ -sulfate). Under these conditions, decomposition of the compound did not actually occur. Increasing the reaction time is expected to increase the yield of chafuroside B. Furthermore, it has been found that even if various impurities coexist in the reaction solution, the reaction efficiency is not substantially affected.
Example 20
鳳凰水仙の茶葉５ｇを、ｐＨ６．４のクエン酸緩衝液を用いてクエン酸／クエン酸ナトリウムの合計濃度が０．６２５、２．５及び１０ｍＭとなるように調整した水溶液各１Ｌを用い、各々常法により抽出した。得られた各抽出液を、プレート式熱交換器を用いて、１４０℃で１分間加熱した。各サンプルを、ＨＰＬＣ−ＭＳ／ＭＳ分析に付し、各成分を定量した。結果を表６に記す。 1 g of each of the aqueous solutions prepared by adjusting 5 g of tea leaves of suisuisen to a total citrate / sodium citrate concentration of 0.625, 2.5 and 10 mM using a citrate buffer of pH 6.4, Extracted by conventional methods. Each obtained extract was heated at 140 ° C. for 1 minute using a plate heat exchanger. Each sample was subjected to HPLC-MS / MS analysis to quantify each component. The results are shown in Table 6.
実施例１９において、反応液中に各種供雑物が存在する場合においても反応が進行することを確認できたので、実施例２０においては、このような供雑物存在下における、添加物の濃度の影響を検証した。すなわち、実施例１９と同様に、出発物質としてチャフロサイドＡ及びＢの前駆体を含有する茶葉抽出液を用いて加熱反応を行った。表６から明らかなように、添加物の終濃度１０ミリモルにおけるチャフロサイドＡ及びＢの収率は、それぞれ５６％、１９％であり、０．６２５ミリモルという低濃度においても１５％、６％という収率を示した。これまでの実施例の結果から、ｐＨ６．４、１４０℃という加熱条件では分解が起こりにくいことから、さらに反応時間を延ばせば、９０％以上の高収率で目的物が得られるであろうことは容易に推察できる。
実施例２１
In Example 19, since it was confirmed that the reaction proceeded even when various contaminants were present in the reaction solution, in Example 20, the concentration of the additive in the presence of such contaminants. The effect of was verified. That is, as in Example 19, a heating reaction was performed using a tea leaf extract containing the precursors of chafuroside A and B as starting materials. As is apparent from Table 6, the yields of chafuroside A and B at a final concentration of 10 mmol of additive were 56% and 19%, respectively, and even at a low concentration of 0.625 mmol, the yields were 15% and 6%. Showed the rate. From the results of the examples so far, decomposition is unlikely to occur under the heating conditions of pH 6.4 and 140 ° C., and if the reaction time is further extended, the target product will be obtained with a high yield of 90% or more. Can be easily guessed.
Example 21
反応液中のチャフロサイドＡ前駆体（イソビテキシン２”−サルフェイト）の濃度が、１μｇ／ｍｌから１２．８ｍｇ／ｍｌとなるように、所定量の水およびクエン酸−リン酸緩衝液を用いてｐＨを６．６に調整した。これらｐＨ調整済水溶液を、マイクロウェーブ加熱装置を用いて、１３０℃で２分間加熱した。得られた各サンプルを、ＨＰＬＣ−ＭＳ／ＭＳ分析に付し、原料のイソビテキシン２”−サルフェイトと生成物であるチャフロサイドＡの含有量を定量した。結果を表７に記す。 Using a predetermined amount of water and citrate-phosphate buffer so that the concentration of chafuroside A precursor (isovitexin 2 ″ -sulfate) in the reaction solution is 1 μg / ml to 12.8 mg / ml. These pH-adjusted aqueous solutions were heated using a microwave heating apparatus for 2 minutes at 130 ° C. Each sample obtained was subjected to HPLC-MS / MS analysis, The contents of isovitexin 2 "-sulfate and the product chafuroside A were quantified. The results are shown in Table 7.
実施例２１は、本発明の製造方法がどのくらい濃い濃度の溶液にまで適用できるのかを確認するために行ったものである。表７から明らかなように、比較的薄い濃度では、原料として用いた仕込み量の増加率と、チャフロサイドＡの生成量の増加率は 必ずしも比例していないが、１２８μｇ／ｍｌ以上の１０倍公比濃度においては、両者の間にきれいな比例関係が成立している。ちなみに、本条件下での最高濃度１２８００μｇ／ｍｌでの反応収率は７１％であった。したがって、試みた最高濃度は１２．８ｍｇ／ｍｌ（１．２８％）であるが、さらに高い濃度、例えば、チャフロサイド類前駆体の１０〜２０％溶液、あるいはそれよりも高い濃度であっても同様の収率で反応が進行することが十分に予想される。 Example 21 was carried out in order to confirm how deep the concentration of the production method of the present invention can be applied. As is clear from Table 7, at a relatively low concentration, the rate of increase in the amount of feed used as a raw material is not necessarily proportional to the rate of increase in the amount of chafuroside A produced, but a 10-fold common ratio of 128 μg / ml or more. A clear proportional relationship is established between the two in terms of concentration. Incidentally, the reaction yield at the maximum concentration of 12800 μg / ml under this condition was 71%. Thus, the highest concentration tried is 12.8 mg / ml (1.28%), but even higher concentrations, such as 10-20% solutions of chafuroside precursors, or higher It is fully expected that the reaction will proceed with a yield of.
以上の実施例の結果から、チャフロサイド類の前駆体である硫酸化体の水溶液を、酸、塩基、塩のいずれか、若しくはそれらの混合物の存在下、ｐＨ５〜１２の範囲において、１００〜１５０℃で加熱処理することにより、チャフロサイド類を操作性よく、効率的かつ簡便に製造する方法を明らかにすることが出来た。そして、本発明における加熱条件については上記以上に特に限定される必要は無く、設備条件、目的に応じて適宜選択する余地が十分にあること、また、反応条件を選べば、９０％以上の極めて高い反応収率が得られることが明らかとなった。 From the results of the above Examples, an aqueous solution of a sulfated body, which is a precursor of chafurosides, is 100 to 150 ° C. in the range of pH 5 to 12 in the presence of any of acid, base, salt, or a mixture thereof. As a result, it was possible to clarify a method for efficiently and easily producing chafurosides with good operability. The heating conditions in the present invention need not be particularly limited as described above, and there is sufficient room for appropriate selection according to the equipment conditions and purpose, and if the reaction conditions are selected, 90% or more of It became clear that a high reaction yield was obtained.
本発明の水溶液中での製造方法により、抗酸化作用、抗アレルギー作用、抗炎症作用、発癌抑制作用を有することが知られているチャフロサイド類を、操作性よく、効率的かつ簡便に製造し提供することができる。
したがって、本製造方法を用いることにより、医薬品用途に供するチャフロサイド類を安価に提供することができるようになる。また、これまで十分に解明されていなかったチャフロサイド類の有用な新たな用途を見出すことが可能となる。さらには、チャフロサイド類の生理活性に基づく、健康を維持するための種々の効果（予防的な効果）を有する製品を提供することができる。
According to the production method in the aqueous solution of the present invention, chafurosides known to have an antioxidative action, an antiallergic action, an anti-inflammatory action, and a carcinogenesis-suppressing action are produced and provided with good operability and efficiency. can do.
Therefore, by using this production method, it is possible to provide chafurosides for pharmaceutical use at low cost. Moreover, it becomes possible to find a useful new use of chafurosides that has not been sufficiently elucidated so far. Furthermore, the product which has various effects (preventive effect) for maintaining health based on the physiological activity of chaflosides can be provided.
Claims (9)
（１）前記硫酸化体を下記の加熱工程に先立ち、酸、塩基、塩のいずれか、若しくはそれらの混合物を含有する水溶液とする工程、
（２）前記工程（１）において、下記加熱工程に付す水溶液のｐＨを５〜１２に調整する工程、および
（３）前記工程（２）を経て得られるｐＨ調整済水溶液を、１００〜１５０℃で加熱処理する工程。 A method for producing chaflosides from a sulfated body which is a precursor of chaflosides, wherein the production method comprises the following steps (1) to (3).
(1) Prior to the following heating step, the sulfated body is an aqueous solution containing any one of an acid, a base, a salt, or a mixture thereof,
(2) In the step (1), the step of adjusting the pH of the aqueous solution to be subjected to the following heating step to 5 to 12, and (3) the pH-adjusted aqueous solution obtained through the step (2) is 100 to 150 ° C. The process of heat-treating with.
9. The production method according to claim 1, wherein the reaction mixture obtained after the heat treatment in the step (3) is further subjected to a purification step to obtain chafuroside A and / or chafuroside B. A method for producing chaflosides.
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