JP2018036214A - Method of isolating and purifying cinnamic acid geometric isomers - Google Patents
Method of isolating and purifying cinnamic acid geometric isomers Download PDFInfo
- Publication number
- JP2018036214A JP2018036214A JP2016171458A JP2016171458A JP2018036214A JP 2018036214 A JP2018036214 A JP 2018036214A JP 2016171458 A JP2016171458 A JP 2016171458A JP 2016171458 A JP2016171458 A JP 2016171458A JP 2018036214 A JP2018036214 A JP 2018036214A
- Authority
- JP
- Japan
- Prior art keywords
- acid
- geometric isomers
- cis
- trans
- cinnamic
- 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
- 235000013985 cinnamic acid Nutrition 0.000 title claims abstract description 38
- WBYWAXJHAXSJNI-UHFFFAOYSA-N methyl p-hydroxycinnamate Natural products OC(=O)C=CC1=CC=CC=C1 WBYWAXJHAXSJNI-UHFFFAOYSA-N 0.000 title claims abstract description 36
- 238000000034 method Methods 0.000 title claims abstract description 28
- WBYWAXJHAXSJNI-VOTSOKGWSA-M .beta-Phenylacrylic acid Natural products [O-]C(=O)\C=C\C1=CC=CC=C1 WBYWAXJHAXSJNI-VOTSOKGWSA-M 0.000 title claims abstract description 23
- WBYWAXJHAXSJNI-SREVYHEPSA-N Cinnamic acid Chemical compound OC(=O)\C=C/C1=CC=CC=C1 WBYWAXJHAXSJNI-SREVYHEPSA-N 0.000 title claims abstract description 23
- 229930016911 cinnamic acid Natural products 0.000 title claims abstract description 23
- NGSWKAQJJWESNS-UHFFFAOYSA-N 4-coumaric acid Chemical compound OC(=O)C=CC1=CC=C(O)C=C1 NGSWKAQJJWESNS-UHFFFAOYSA-N 0.000 claims abstract description 38
- 238000004811 liquid chromatography Methods 0.000 claims abstract description 23
- 239000003443 antiviral agent Substances 0.000 claims abstract description 21
- 239000000203 mixture Substances 0.000 claims abstract description 21
- 229920002678 cellulose Polymers 0.000 claims abstract description 18
- 239000003125 aqueous solvent Substances 0.000 claims abstract description 10
- 239000003960 organic solvent Substances 0.000 claims abstract description 10
- 238000000746 purification Methods 0.000 claims abstract description 10
- 230000005526 G1 to G0 transition Effects 0.000 claims abstract description 9
- 230000002378 acidificating effect Effects 0.000 claims abstract description 9
- NGSWKAQJJWESNS-UTCJRWHESA-N cis-4-coumaric acid Chemical compound OC(=O)\C=C/C1=CC=C(O)C=C1 NGSWKAQJJWESNS-UTCJRWHESA-N 0.000 claims description 16
- 238000000926 separation method Methods 0.000 claims description 14
- 239000001913 cellulose Substances 0.000 claims description 12
- 239000003814 drug Substances 0.000 claims description 11
- 229940079593 drug Drugs 0.000 claims description 11
- 230000000840 anti-viral effect Effects 0.000 claims description 7
- 239000012046 mixed solvent Substances 0.000 claims description 6
- 239000004480 active ingredient Substances 0.000 claims description 5
- NGSWKAQJJWESNS-ZZXKWVIFSA-M 4-Hydroxycinnamate Natural products OC1=CC=C(\C=C\C([O-])=O)C=C1 NGSWKAQJJWESNS-ZZXKWVIFSA-M 0.000 abstract description 28
- QAIPRVGONGVQAS-DUXPYHPUSA-N trans-caffeic acid Chemical compound OC(=O)\C=C\C1=CC=C(O)C(O)=C1 QAIPRVGONGVQAS-DUXPYHPUSA-N 0.000 abstract description 22
- 235000010980 cellulose Nutrition 0.000 abstract description 17
- ACEAELOMUCBPJP-UHFFFAOYSA-N (E)-3,4,5-trihydroxycinnamic acid Natural products OC(=O)C=CC1=CC(O)=C(O)C(O)=C1 ACEAELOMUCBPJP-UHFFFAOYSA-N 0.000 abstract description 11
- KSEBMYQBYZTDHS-HWKANZROSA-M (E)-Ferulic acid Natural products COC1=CC(\C=C\C([O-])=O)=CC=C1O KSEBMYQBYZTDHS-HWKANZROSA-M 0.000 abstract description 11
- DFYRUELUNQRZTB-UHFFFAOYSA-N Acetovanillone Natural products COC1=CC(C(C)=O)=CC=C1O DFYRUELUNQRZTB-UHFFFAOYSA-N 0.000 abstract description 11
- 235000004883 caffeic acid Nutrition 0.000 abstract description 11
- 229940074360 caffeic acid Drugs 0.000 abstract description 11
- QAIPRVGONGVQAS-UHFFFAOYSA-N cis-caffeic acid Natural products OC(=O)C=CC1=CC=C(O)C(O)=C1 QAIPRVGONGVQAS-UHFFFAOYSA-N 0.000 abstract description 11
- KSEBMYQBYZTDHS-HWKANZROSA-N ferulic acid Chemical compound COC1=CC(\C=C\C(O)=O)=CC=C1O KSEBMYQBYZTDHS-HWKANZROSA-N 0.000 abstract description 11
- 235000001785 ferulic acid Nutrition 0.000 abstract description 11
- 229940114124 ferulic acid Drugs 0.000 abstract description 11
- KSEBMYQBYZTDHS-UHFFFAOYSA-N ferulic acid Natural products COC1=CC(C=CC(O)=O)=CC=C1O KSEBMYQBYZTDHS-UHFFFAOYSA-N 0.000 abstract description 11
- QURCVMIEKCOAJU-UHFFFAOYSA-N trans-isoferulic acid Natural products COC1=CC=C(C=CC(O)=O)C=C1O QURCVMIEKCOAJU-UHFFFAOYSA-N 0.000 abstract description 11
- PCMORTLOPMLEFB-ONEGZZNKSA-N sinapic acid Chemical compound COC1=CC(\C=C\C(O)=O)=CC(OC)=C1O PCMORTLOPMLEFB-ONEGZZNKSA-N 0.000 abstract description 4
- PCMORTLOPMLEFB-UHFFFAOYSA-N sinapinic acid Natural products COC1=CC(C=CC(O)=O)=CC(OC)=C1O PCMORTLOPMLEFB-UHFFFAOYSA-N 0.000 abstract description 3
- 239000002904 solvent Substances 0.000 abstract description 3
- 238000002955 isolation Methods 0.000 abstract 1
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 24
- NGSWKAQJJWESNS-ZZXKWVIFSA-N trans-4-coumaric acid Chemical compound OC(=O)\C=C\C1=CC=C(O)C=C1 NGSWKAQJJWESNS-ZZXKWVIFSA-N 0.000 description 18
- 241000700605 Viruses Species 0.000 description 14
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 12
- 239000000243 solution Substances 0.000 description 9
- 238000004128 high performance liquid chromatography Methods 0.000 description 8
- ZVQOOHYFBIDMTQ-UHFFFAOYSA-N [methyl(oxido){1-[6-(trifluoromethyl)pyridin-3-yl]ethyl}-lambda(6)-sulfanylidene]cyanamide Chemical compound N#CN=S(C)(=O)C(C)C1=CC=C(C(F)(F)F)N=C1 ZVQOOHYFBIDMTQ-UHFFFAOYSA-N 0.000 description 7
- DYLIWHYUXAJDOJ-OWOJBTEDSA-N (e)-4-(6-aminopurin-9-yl)but-2-en-1-ol Chemical compound NC1=NC=NC2=C1N=CN2C\C=C\CO DYLIWHYUXAJDOJ-OWOJBTEDSA-N 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 230000002779 inactivation Effects 0.000 description 6
- 238000004809 thin layer chromatography Methods 0.000 description 6
- 241000712431 Influenza A virus Species 0.000 description 4
- 238000005481 NMR spectroscopy Methods 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 208000015181 infectious disease Diseases 0.000 description 4
- 230000002458 infectious effect Effects 0.000 description 4
- 150000007524 organic acids Chemical class 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- -1 Cinnamic acids Cinnamic acids Chemical class 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 239000007864 aqueous solution Substances 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 239000011521 glass Substances 0.000 description 3
- 239000002608 ionic liquid Substances 0.000 description 3
- 230000014759 maintenance of location Effects 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 150000007522 mineralic acids Chemical class 0.000 description 3
- 239000002953 phosphate buffered saline Substances 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- IAZDPXIOMUYVGZ-WFGJKAKNSA-N Dimethyl sulfoxide Chemical compound [2H]C([2H])([2H])S(=O)C([2H])([2H])[2H] IAZDPXIOMUYVGZ-WFGJKAKNSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 239000003963 antioxidant agent Substances 0.000 description 2
- 230000003078 antioxidant effect Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 239000007979 citrate buffer Substances 0.000 description 2
- 239000002537 cosmetic Substances 0.000 description 2
- 239000002781 deodorant agent Substances 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 150000002148 esters Chemical class 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000012091 fetal bovine serum Substances 0.000 description 2
- 239000000796 flavoring agent Substances 0.000 description 2
- 235000013355 food flavoring agent Nutrition 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 239000002324 mouth wash Substances 0.000 description 2
- 239000000820 nonprescription drug Substances 0.000 description 2
- 239000000825 pharmaceutical preparation Substances 0.000 description 2
- 229940127557 pharmaceutical product Drugs 0.000 description 2
- 150000002989 phenols Chemical class 0.000 description 2
- 239000003755 preservative agent Substances 0.000 description 2
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000003381 stabilizer Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000006188 syrup Substances 0.000 description 2
- 235000020357 syrup Nutrition 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- AAWZDTNXLSGCEK-LNVDRNJUSA-N (3r,5r)-1,3,4,5-tetrahydroxycyclohexane-1-carboxylic acid Chemical compound O[C@@H]1CC(O)(C(O)=O)C[C@@H](O)C1O AAWZDTNXLSGCEK-LNVDRNJUSA-N 0.000 description 1
- PFRQBZFETXBLTP-RCIYGOBDSA-N 2-[(2e,6e,10e,14e,18e)-3,7,11,15,19,23-hexamethyltetracosa-2,6,10,14,18,22-hexaen-1-yl]-3-methyl-1,4-dihydronaphthalene-1,4-dione Chemical compound C1=CC=C2C(=O)C(C/C=C(C)/CC/C=C(C)/CC/C=C(C)/CC/C=C(C)/CC/C=C(C)/CCC=C(C)C)=C(C)C(=O)C2=C1 PFRQBZFETXBLTP-RCIYGOBDSA-N 0.000 description 1
- CWVRJTMFETXNAD-FWCWNIRPSA-N 3-O-Caffeoylquinic acid Natural products O[C@H]1[C@@H](O)C[C@@](O)(C(O)=O)C[C@H]1OC(=O)\C=C\C1=CC=C(O)C(O)=C1 CWVRJTMFETXNAD-FWCWNIRPSA-N 0.000 description 1
- 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 1
- LTUMRKDLVGQMJU-UHFFFAOYSA-N 6,10,14-trimethylpentadeca-5,9,13-trien-2-one Chemical compound CC(C)=CCCC(C)=CCCC(C)=CCCC(C)=O LTUMRKDLVGQMJU-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 210000003771 C cell Anatomy 0.000 description 1
- PZIRUHCJZBGLDY-UHFFFAOYSA-N Caffeoylquinic acid Natural products CC(CCC(=O)C(C)C1C(=O)CC2C3CC(O)C4CC(O)CCC4(C)C3CCC12C)C(=O)O PZIRUHCJZBGLDY-UHFFFAOYSA-N 0.000 description 1
- 241000282465 Canis Species 0.000 description 1
- AAWZDTNXLSGCEK-UHFFFAOYSA-N Cordycepinsaeure Natural products OC1CC(O)(C(O)=O)CC(O)C1O AAWZDTNXLSGCEK-UHFFFAOYSA-N 0.000 description 1
- 239000006145 Eagle's minimal essential medium Substances 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
- 244000017020 Ipomoea batatas Species 0.000 description 1
- 235000002678 Ipomoea batatas Nutrition 0.000 description 1
- 238000003820 Medium-pressure liquid chromatography Methods 0.000 description 1
- 229920000168 Microcrystalline cellulose Polymers 0.000 description 1
- CWVRJTMFETXNAD-KLZCAUPSSA-N Neochlorogenin-saeure Natural products O[C@H]1C[C@@](O)(C[C@@H](OC(=O)C=Cc2ccc(O)c(O)c2)[C@@H]1O)C(=O)O CWVRJTMFETXNAD-KLZCAUPSSA-N 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 101710141175 Photoactive yellow protein Proteins 0.000 description 1
- AAWZDTNXLSGCEK-ZHQZDSKASA-N Quinic acid Natural products O[C@H]1CC(O)(C(O)=O)C[C@H](O)C1O AAWZDTNXLSGCEK-ZHQZDSKASA-N 0.000 description 1
- 230000006750 UV protection Effects 0.000 description 1
- 238000011481 absorbance measurement Methods 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 230000003276 anti-hypertensive effect Effects 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 230000036772 blood pressure Effects 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 235000001368 chlorogenic acid Nutrition 0.000 description 1
- CWVRJTMFETXNAD-JUHZACGLSA-N chlorogenic acid Chemical compound O[C@@H]1[C@H](O)C[C@@](O)(C(O)=O)C[C@H]1OC(=O)\C=C\C1=CC=C(O)C(O)=C1 CWVRJTMFETXNAD-JUHZACGLSA-N 0.000 description 1
- 229940074393 chlorogenic acid Drugs 0.000 description 1
- FFQSDFBBSXGVKF-KHSQJDLVSA-N chlorogenic acid Natural products O[C@@H]1C[C@](O)(C[C@@H](CC(=O)C=Cc2ccc(O)c(O)c2)[C@@H]1O)C(=O)O FFQSDFBBSXGVKF-KHSQJDLVSA-N 0.000 description 1
- BMRSEYFENKXDIS-KLZCAUPSSA-N cis-3-O-p-coumaroylquinic acid Natural products O[C@H]1C[C@@](O)(C[C@@H](OC(=O)C=Cc2ccc(O)cc2)[C@@H]1O)C(=O)O BMRSEYFENKXDIS-KLZCAUPSSA-N 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 229940124579 cold medicine Drugs 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000000645 desinfectant Substances 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 235000015872 dietary supplement Nutrition 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000007884 disintegrant Substances 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 229930005346 hydroxycinnamic acid Natural products 0.000 description 1
- 235000010359 hydroxycinnamic acids Nutrition 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 238000003819 low-pressure liquid chromatography Methods 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 235000019813 microcrystalline cellulose Nutrition 0.000 description 1
- 239000008108 microcrystalline cellulose Substances 0.000 description 1
- 229940016286 microcrystalline cellulose Drugs 0.000 description 1
- 239000007758 minimum essential medium Substances 0.000 description 1
- 238000004264 monolayer culture Methods 0.000 description 1
- 229940051866 mouthwash Drugs 0.000 description 1
- 239000002674 ointment Substances 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 230000000243 photosynthetic effect Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 239000012265 solid product Substances 0.000 description 1
- 239000010902 straw Substances 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- WROMPOXWARCANT-UHFFFAOYSA-N tfa trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F.OC(=O)C(F)(F)F WROMPOXWARCANT-UHFFFAOYSA-N 0.000 description 1
- 125000000391 vinyl group Chemical group [H]C([*])=C([H])[H] 0.000 description 1
Landscapes
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
- Treatment Of Liquids With Adsorbents In General (AREA)
Abstract
Description
本発明は、桂皮酸類幾何異性体の分離精製法等に関する。 The present invention relates to a method for separating and purifying cinnamic acid geometric isomers and the like.
桂皮酸、p-クマル酸、カフェ酸、フェルラ酸などの桂皮酸類は、フェノール性化合物の一種であり、これら有機酸と糖とが主にエステル結合した状態で、植物や微生物の二次代謝産物として広く分布している。 Cinnamic acid, such as cinnamic acid, p-coumaric acid, caffeic acid, and ferulic acid, is a type of phenolic compound, and is a secondary metabolite of plants and microorganisms in the state that these organic acids and sugars are mainly ester-linked. As widely distributed.
例えば、コーヒーやサツマイモなどに存在するクロロゲン酸は、カフェ酸とキナ酸がエステル結合したものである。また、米糠や麦わらにはフェルラ酸が多糖類と結合して大量に存在している。 For example, chlorogenic acid present in coffee, sweet potato, etc. is an ester-linked caffeic acid and quinic acid. In addition, ferulic acid is present in large amounts in rice bran and straw combined with polysaccharides.
なお、これらの桂皮酸類は、抗酸化作用や降圧作用などの作用を有し、食品の酸化防止や紫外線防止の化粧品や血圧対策のための特定保健用食品などとして広く利用されている。また、p-クマル酸は、植物フェノール化合物の生合成の制御やアレロパシーに関与したり、紅色光合成細菌のPhotoactive Yellow Protein（Hh-PYP）の発色団としての役割も報告されているため、生物学的研究の対照としても重要である。 These cinnamic acids have antioxidative and antihypertensive effects, and are widely used as foods for antioxidants and UV protection cosmetics, foods for specified health use for blood pressure countermeasures, and the like. In addition, p-coumaric acid has been reported to play a role in the control and biosynthesis of plant phenolic compounds and as a chromophore of the photoactive yellow protein (Hh-PYP) of red photosynthetic bacteria. It is also important as a control for research.
桂皮酸類は、trans体とcis体の幾何異性体を含んでいる。このうち、桂皮酸類のtrans体は、化学合成や植物材料などから容易に入手できるものの、cis体の入手は容易ではなかった。例えば、p-クマル酸の場合、trans体は化学合成により容易に入手できるが、充分な量のcis体を化学合成によって入手することは従来極めて困難であった。 Cinnamic acids include trans and cis geometric isomers. Among these, cinnamic acid trans isomers can be easily obtained from chemical synthesis or plant materials, but cis isomers have not been readily available. For example, in the case of p-coumaric acid, the trans isomer can be easily obtained by chemical synthesis, but it has been extremely difficult to obtain a sufficient amount of cis isomer by chemical synthesis.
従来、桂皮酸類のcis体は、（１）桂皮酸類のtrans体を紫外線処理してtrans体からcis体が生成させ、一定時間後にtrans体とcis体とを化学量的に平衡状態にする、（２）平衡状態にあるtrans体とcis体とを分離する方法により入手されている。なお、（２）では、イオン液体を使用する異性体分離法でtrans体とcis体とを分離したのち、異性体の溶解度の差を使用して各幾何異性体を精製している（非特許文献１を参照。）。 Conventionally, the cis form of cinnamic acids is (1) the trans form of cinnamic acids is treated with ultraviolet rays to form a cis form from the trans form, and after a certain period of time, the trans form and the cis form are stoichiometrically balanced (2) Obtained by a method of separating the trans and cis bodies in equilibrium. In (2), the trans isomer and cis isomer are separated by an isomer separation method using an ionic liquid, and then the geometric isomers are purified using the difference in solubility of the isomers (non-patent) See reference 1.)
このように、従来からある製造方法では、非常に高価なイオン液体を使用しなければならず、製造規模に応じてイオン液体の使用量を増加させなければならないため、桂皮酸類のcis体を大量、かつ安価に入手することは困難であった。そのため、例えばp-クマル酸のcis体は、非常に高価（100mgで12万円）であり、例えば、抗ウイルス剤などの実用的な用途に使用することはできなかった。 In this way, in the conventional production method, a very expensive ionic liquid must be used, and the amount of ionic liquid used must be increased according to the production scale. In addition, it was difficult to obtain them at low cost. Therefore, for example, the cis form of p-coumaric acid is very expensive (120,000 yen at 100 mg) and could not be used for practical applications such as antiviral agents.
一方、ビタミンK2に含まれる幾何異性体のうちのtrans体を、臨界流体クロマトグラフィーを使用して工業的、すなわち大量かつ安価に分離精製する方法が、既に知られている（特許文献１を参照。）。また、6,10,14-トリメチルペンタデカ-5,9,13−トリエン-2-オンの幾何異性体混合物を、液体クロマトグラフィーにより幾何異性体混合ごとに分離する方法が、既に知られている（特許文献２を参照。）。 On the other hand, a method for separating and purifying trans isomers of geometric isomers contained in vitamin K 2 industrially, that is, in a large amount and at low cost, using critical fluid chromatography is already known (see Patent Document 1). reference.). Further, a method for separating a geometric isomer mixture of 6,10,14-trimethylpentadeca-5,9,13-trien-2-one for each geometric isomer mixture by liquid chromatography is already known. (See Patent Document 2).
本発明は、桂皮酸、p-クマル酸、カフェ酸、フェルラ酸、シナピン酸などの桂皮酸類の幾何異性体混合物から、各幾何異性体を大量かつ安価に分離精製する方法を提供することを課題とする。また、分離精製した桂皮酸類のcis体のみを含む抗ウイルス剤などを提供することも課題とする。 An object of the present invention is to provide a method for separating and purifying each geometric isomer in a large amount and at low cost from a mixture of geometric isomers of cinnamic acids such as cinnamic acid, p-coumaric acid, caffeic acid, ferulic acid, and sinapinic acid. And Another object of the present invention is to provide an antiviral agent containing only the cis form of cinnamic acid that has been separated and purified.
発明者らは、鋭意検討の結果、セルロースカラムを使用する液体クロマトグラフィーによって、桂皮酸類幾何異性体の混合物をcis体とtrans体に分離精製できることを見出し、本発明を完成させた。また、分離精製した桂皮酸類のcis体が抗ウイルス性を有することを見出し、本発明を完成させた。 As a result of intensive studies, the inventors have found that a mixture of cinnamic acid geometric isomers can be separated and purified into a cis form and a trans form by liquid chromatography using a cellulose column, and completed the present invention. In addition, the present inventors have found that the cis form of cinnamic acid that has been separated and purified has antiviral properties and completed the present invention.
すなわち、本発明は、桂皮酸類の幾何異性体混合物から液体クロマトグラフィーによって幾何異性体を分離精製する分離精製法であって、固定相にセルロース類を使用する方法である。 That is, the present invention is a separation and purification method for separating and purifying geometric isomers from a mixture of geometric isomers of cinnamic acids by liquid chromatography, and using celluloses as a stationary phase.
また、本発明は、固定相にセルロース類を使用するとともに、移動相に酸性水性溶媒と有機溶媒との混合溶媒を使用する分離精製法である。 The present invention is also a separation and purification method using celluloses as a stationary phase and a mixed solvent of an acidic aqueous solvent and an organic solvent as a mobile phase.
さらに、本発明は、桂皮酸類、中でもp-クマル酸の幾何異性体のうちcis体のみを有効成分として含む抗ウイルス剤である。加えて、本発明は前記抗ウイルス剤を含む医薬品又は医薬部外品である。 Furthermore, the present invention is an antiviral agent comprising, as an active ingredient, only cinnamic acid, especially cis isomer of p-coumaric acid. In addition, the present invention is a pharmaceutical or quasi drug containing the antiviral agent.
本発明の桂皮酸類幾何異性体の分離精製法は、生化学分野では一般的な液体クロマトグラフィーを使用して桂皮酸類を幾何異性体ごとに分離精製するので、桂皮酸類のcis体のみを大量かつ安価に入手できる。 In the method of separating and purifying cinnamic acid geometric isomers of the present invention, cinnamic acids are separated and purified by geometric isomers using liquid chromatography, which is common in the biochemical field. It can be obtained inexpensively.
また、本発明の抗ウイルス剤は、桂皮酸類のcis体のみを有効成分として含んでいるため、優れた抗ウイルス効果を有している。本発明の医薬品又は医薬部外品は、本発明の抗ウイルス剤を含んでいるため、優れた抗ウイルス効果を有している。 Moreover, since the antiviral agent of the present invention contains only the cis form of cinnamic acid as an active ingredient, it has an excellent antiviral effect. Since the pharmaceutical or quasi drug of the present invention contains the antiviral agent of the present invention, it has an excellent antiviral effect.
本発明の桂皮酸類の分離精製法は、桂皮酸類の幾何異性体混合物から液体クロマトグラフィーによって幾何異性体を分離精製する分離精製法であって、固定相にセルロース類を使用する方法である。そこで、本発明について以下に詳説する。 The method for separating and purifying cinnamic acids according to the present invention is a method for separating and purifying geometric isomers from a mixture of geometric isomers of cinnamic acids by liquid chromatography, which uses celluloses as a stationary phase. Therefore, the present invention will be described in detail below.
（１）桂皮酸類
本発明で分離精製する桂皮酸類とは、桂皮酸及びその誘導体、具体的には、ヒドロキシ桂皮酸であるp-クマル酸、カフェ酸、フェルラ酸、シナピン酸が挙げられる。
(1) Cinnamic acids Cinnamic acids to be separated and purified in the present invention include cinnamic acid and its derivatives, specifically, p-coumaric acid, which is hydroxycinnamic acid, caffeic acid, ferulic acid, and sinapinic acid.
（２）液体クロマトグラフィー
本発明の分離精製法における液体クロマトグラフィーは、公知の液体クロマトグラフィー方法であれば特に限定することなく使用できる。具体的には、高速液体クロマトグラフィー法（HPLC）、中圧液体クロマトグラフィー法、低圧液体クロマトグラフィーが使用できる。なお、これらのクロマトグラフィー法において、単カラム法はもちろん、多段カラム法、擬似移動床法及び改良型擬似移動床法等を使用してもよい。
(2) Liquid Chromatography Liquid chromatography in the separation and purification method of the present invention can be used without particular limitation as long as it is a known liquid chromatography method. Specifically, high performance liquid chromatography (HPLC), medium pressure liquid chromatography, and low pressure liquid chromatography can be used. In these chromatography methods, a single column method, a multistage column method, a simulated moving bed method, an improved simulated moving bed method, and the like may be used.
（３）固定相
液体クロマトグラフィーで使用する固定相は、セルロース類である。ここで、セルロース類とは、セルロース又はその誘導体のことであり、液体クロマトグラフィーで使用されている公知のセルロース又はその誘導体であれば特に限定することなく使用できる。なお、セルロース誘導体としては、水酸基の水素原子がカルボニル基、ビニル基により置換されたセルロース誘導体が例示できる。
(3) Stationary phase The stationary phase used in liquid chromatography is celluloses. Here, the cellulose refers to cellulose or a derivative thereof, and any known cellulose or derivative thereof used in liquid chromatography can be used without particular limitation. In addition, as a cellulose derivative, the cellulose derivative by which the hydrogen atom of the hydroxyl group was substituted by the carbonyl group and the vinyl group can be illustrated.
（４）移動相
液体クロマトグラフィーで使用する移動相は、酸性水性溶媒と親水性の有機溶媒の混合溶媒である。酸性に保つことで桂皮酸類の溶解度を増し、安定性を強める。また親水性の有機溶媒を含むことにより、桂皮酸の溶解度を増強することができる。なお、移動相は、この他に必要に応じて、酸化防止剤などを含んでいてもよい。
(4) Mobile phase The mobile phase used in liquid chromatography is a mixed solvent of an acidic aqueous solvent and a hydrophilic organic solvent. Maintaining acidity increases cinnamic acid solubility and enhances stability. Moreover, the solubility of cinnamic acid can be enhanced by including a hydrophilic organic solvent. In addition, the mobile phase may contain an antioxidant or the like as necessary.
１）酸性水性溶媒
酸性水性溶媒は、桂皮酸類を可溶な公知のギ酸、酢酸、プロピオンなどの有機酸又はリン酸、塩酸などの無機酸を含む酸性水性溶媒であれば限定することなく使用できる。なお、酸性水性溶媒は、一種類の有機酸又は無機酸だけを含んでいてもよく、複数種の有機酸又は無機酸を含んでいてもよい
1) Acidic aqueous solvent The acidic aqueous solvent can be used without limitation as long as it is an acidic aqueous solvent containing a known organic acid such as formic acid, acetic acid, propion or the like, or inorganic acid such as phosphoric acid or hydrochloric acid which is soluble in cinnamic acid. . The acidic aqueous solvent may contain only one type of organic acid or inorganic acid, or may contain a plurality of types of organic acid or inorganic acid.
２）有機溶媒
有機溶媒としては、桂皮酸類を可溶で水性溶媒と混合可能な公知の有機溶媒であれば特に限定することなく使用できる。具体的には、例えば、メタノール、エタノール、イソプロパノール、n-プロパノールなどの低級アルコールや、アセトン、アセトニトリルなどが挙げられる。なお、これら有機溶媒は、単独で使用してもよく、複数を組み合わせて使用してもよい。
2) Organic solvent Any organic solvent may be used without particular limitation as long as it is a known organic solvent that is soluble in cinnamic acid and can be mixed with an aqueous solvent. Specific examples include lower alcohols such as methanol, ethanol, isopropanol, and n-propanol, acetone, and acetonitrile. These organic solvents may be used alone or in combination.
このように、本発明の分離精製法は、液体クロマトグラフィーの固定相として生化学分野において一般なセルロース類を使用し、移動相に生化学分野において一般な水性溶媒と有機溶媒の混合溶媒を使用する。そのため、従来方法とは異なり、大量かつ安価に桂皮酸類の幾何異性体を分離精製できる。 Thus, in the separation and purification method of the present invention, celluloses that are common in the biochemical field are used as the stationary phase of liquid chromatography, and a mixed solvent of an aqueous solvent and an organic solvent that is common in the biochemical field is used as the mobile phase. To do. Therefore, unlike conventional methods, cinnamic acid geometric isomers can be separated and purified in large quantities and at low cost.
２．抗ウイルス剤
本発明の抗ウイルス剤は、本発明の分離精製法によって大量かつ安価に得られるようになった桂皮酸類のcis体のみを有効成分として含むものである。
2. Antiviral Agent The antiviral agent of the present invention contains only the cis form of cinnamic acids that can be obtained in large quantities and at low cost by the separation and purification method of the present invention as an active ingredient.
３．抗ウイルス用医薬品及び抗ウイルス用医薬部外品
本発明の医薬品又は医薬部外品は、本発明の抗ウイルス剤を含むものである。なお、医薬品及び医薬部外品における抗ウイルス剤の含有量は、使用する抗ウイルス剤の抗ウイルス力やその用途等を勘案して自由に設定することができる。
3. Antiviral drug and antiviral quasi-drug The pharmaceutical or quasi-drug of the present invention contains the antiviral agent of the present invention. In addition, content of the antiviral agent in a pharmaceutical and a quasi-drug can be freely set in consideration of the antiviral power of the antiviral agent to be used, its use, and the like.
（１）医薬品
医薬品とは、薬事法に規定されているものであって、医療用医薬品及び一般用医薬品（OTC）の何れをも含む。また、その対象となる疾患は従来からある抗ウイルス剤を含む医薬品、例えば風邪薬、整腸剤、含嗽薬などであれば、特に限定することなく使用できる。さらに、その形態については、例えば、丸薬剤、液剤、粉末剤、顆粒剤、錠剤、カプセル錠剤、トローチ剤、シロップ剤、ドライシロップ剤、懸濁液、エマルジョン剤、エリキシル剤などの経口剤、注射剤、坐剤、外用液剤、軟膏等の塗布剤等の非経口剤などが挙げられるが、これらに限定されるものではない。
(1) Drugs Drugs are stipulated in the Pharmaceutical Affairs Law and include both ethical drugs and over-the-counter drugs (OTC). Moreover, if the disease used as the object is a pharmaceutical including a conventional antiviral agent, for example, a cold medicine, an intestinal preparation, a mouthwash, etc., it can be used without particular limitation. In addition, for example, pills, liquids, powders, granules, tablets, capsule tablets, troches, syrups, dry syrups, suspensions, emulsions, elixirs and other oral preparations, injections And non-oral agents such as suppositories, liquids for external use, and coating agents such as ointments, but are not limited thereto.
なお、本発明の医薬品を経口剤として製造する場合には、公知の賦型剤、結合剤、崩壊剤、界面活性剤、滑沢剤、流動性促進剤、矯味剤、矯臭剤、着色剤等とともに、公知の製造方法により製造すればよい。 In the case of producing the pharmaceutical product of the present invention as an oral preparation, known excipients, binders, disintegrants, surfactants, lubricants, fluidity promoters, flavoring agents, flavoring agents, coloring agents, etc. At the same time, it may be manufactured by a known manufacturing method.
また、本発明の医薬品を非経口剤として製造する場合には、注射用蒸留水、生理食塩水希釈剤、ブドウ糖水溶液等の希釈剤、公知の殺菌剤、防腐剤、安定剤、等張化剤、安定剤、防腐剤、無痛化剤とともに、公知の方法によって製造すればよい。 Further, when the pharmaceutical product of the present invention is produced as a parenteral agent, diluents such as distilled water for injection, physiological saline diluent, aqueous glucose solution, known disinfectants, preservatives, stabilizers, tonicity agents In addition to the stabilizer, preservative, and soothing agent, it may be produced by a known method.
（２）医薬部外品
医薬部外品とは、薬事法に規定されているものであって、例えば、含嗽剤、脱臭剤（デオドラント剤）、育毛剤、薬用化粧品類、栄養補給薬（サプリメント）等が挙げられるが、これらに限定されるものではない。
(2) Quasi-drugs Quasi-drugs are stipulated in the Pharmaceutical Affairs Law and include, for example, mouthwashes, deodorants (deodorants), hair restorers, medicated cosmetics, nutritional supplements (supplements) ) And the like, but are not limited thereto.
以下、本発明について実施例に基づいてより詳細に説明する。なお、本発明の特許請求の範囲は、以下の実施例によって如何なる意味においても制限されない。 Hereinafter, the present invention will be described in more detail based on examples. The claims of the present invention are not limited in any way by the following examples.
本発明の分離精製法に従って、桂皮酸類幾何異性体の混合物をcis体とtrans体に分離精製し、その分離能力を評価した。以下に詳説する。 According to the separation and purification method of the present invention, a mixture of cinnamic acid geometric isomers was separated and purified into a cis form and a trans form, and the separation ability was evaluated. The details are described below.
１.試薬及び実験装置等
（１）試薬
trans-p-クマル酸（SIGMA-ALDRICH）
cis-p-クマル酸（Tronto Research Chemicals）
カフェ酸（SIGMA-ALDRICH）
フェルラ酸（築野食品工業株式会社）
1. Reagents and experimental equipment (1) Reagents
trans-p-coumaric acid (SIGMA-ALDRICH)
cis-p-Coumaric acid (Tronto Research Chemicals)
Caffeic acid (SIGMA-ALDRICH)
Ferulic acid (Tsukino Food Industry Co., Ltd.)
（２）実験装置及び測定条件
１）紫外線ランプ（Sankyo G15F8E、三共電気株式会社）
２）液体クロマトグラフィー
カラム ガラスカラム（直径50 mm×長さ300 mm、株式会社旭製作所）
固定相 微結晶セルロース（Merck社）
移動相 1容量%酢酸水溶液：メタノール(=90:10,v/v)の混合溶媒
検出 吸光度（測定波長：280nm）
３）HPLC
HPLC装置 Prominence HPLC system （株式会社島津製作所）
分離用カラム Hydrosphere C18 250 mm×4.6 mm I.D. （5μm、株式会社ワイエムシィ）
ガードカラム InertSustain C18 カートリッジガードカラムE 10 mm×4.0 mm I.D. （5μm、ジーエルサイエンス株式会社）
カラム温度 30 ℃
セル部温調温度 40 ℃
移動相A 0.1％トリフルオロ酢酸（TFA）水溶液
移動相B メタノール
流速 1.0 mL/ min
検出 吸光度（測定波長：280 nm）
グラジエント条件
時間（min.） 移動相Bの濃度（容量％）
0 20
10 20
80 75
80.01-90.00 100
90.01-105.00 20
４）薄層クロマトグラフィー（TLC）
TLCセルロースプレート(メルク株式会社、カタログ番号 105716)
展開溶媒 10容量%メタノール+1容量％酢酸水溶液
５）NMR
NMR装置 JNM-ECA 400 FT NMR(株式会社 JEOL RESONANCE)
溶媒 DMSO-d6
(2) Experimental equipment and measurement conditions 1) Ultraviolet lamp (Sankyo G15F8E, Sankyo Electric Co., Ltd.)
2) Liquid chromatography column Glass column (diameter 50 mm x length 300 mm, Asahi Seisakusho Co., Ltd.)
Stationary phase Microcrystalline cellulose (Merck)
Mobile phase 1% by volume acetic acid aqueous solution: methanol (= 90: 10, v / v) mixed solvent Detection Absorbance (measurement wavelength: 280 nm)
3) HPLC
HPLC system Prominence HPLC system (Shimadzu Corporation)
Separation column Hydrosphere C18 250 mm x 4.6 mm ID (5μm, YMC Co., Ltd.)
Guard column InertSustain C18 Cartridge guard column E 10 mm x 4.0 mm ID (5μm, GL Sciences Inc.)
Column temperature 30 ° C
Cell temperature control temperature 40 ℃
Mobile phase A 0.1% trifluoroacetic acid (TFA) aqueous solution Mobile phase B Methanol Flow rate 1.0 mL / min
Absorbance (measurement wavelength: 280 nm)
Gradient condition Time (min.) Concentration of mobile phase B (volume%)
0 20
10 20
80 75
80.01-90.00 100
90.01-105.00 20
4) Thin layer chromatography (TLC)
TLC cellulose plate (Merck, catalog number 105716)
Developing solvent 10% by volume methanol + 1% by volume acetic acid aqueous solution
5) NMR
NMR system JNM-ECA 400 FT NMR (JEOL RESONANCE Co., Ltd.)
Solvent DMSO-d6
２．実験方法と実験結果
（１）混合物の調製
trans-p-クマル酸を100%メタノールに溶解し（1000μg/mL）、ガラスバイアル（20.3×40.0×75mm）に入れた。ガラスバイアルの外から、0.75 mW/cm2±10％の強度の紫外線を72時間照射した。HPLC及びTLCによって、cis体が生成し、cis体とtrans体の混合物となっていることを確認した（データは示さない。）。
2. Experimental methods and results (1) Preparation of the mixture
trans-p-coumaric acid was dissolved in 100% methanol (1000 μg / mL) and placed in a glass vial (20.3 × 40.0 × 75 mm). Ultraviolet rays with an intensity of 0.75 mW / cm 2 ± 10% were irradiated for 72 hours from the outside of the glass vial. It was confirmed by HPLC and TLC that a cis isomer was produced and a mixture of cis isomer and trans isomer was obtained (data not shown).
（２）液体クロマトグラフィーによる分離
混合物のメタノール溶液を、ロータリーエバポレーターで蒸発乾固させた。乾固物をもとの5分の1のメタノールに溶解して、分離用試料とした。セルロースカラムに試料を5mL注ぎ、移動相である酢酸とメタノールの混合溶媒で溶出し、溶出液を5mLずつ分取した。なお、分取する際に各画分の吸光度を測定し、画分ごとの吸光度の測定結果をチャートにした。その結果を図１に示す。
(2) Separation by liquid chromatography The methanol solution of the mixture was evaporated to dryness on a rotary evaporator. The dried product was dissolved in 1/5 methanol to obtain a sample for separation. 5 mL of the sample was poured onto a cellulose column and eluted with a mixed solvent of acetic acid and methanol as a mobile phase, and 5 mL of the eluate was fractionated. The absorbance of each fraction was measured at the time of fractionation, and the absorbance measurement results for each fraction were charted. The result is shown in FIG.
図１に示すように、チャートには前側と後側の２つのピークがある、すなわち、混合物がcis体とtrans体の２つの幾何異性体の混合物であることが分かった。そこで、各ピークが含む物質を同定するため、前側ピークの画分を集め、ロータリーエバポレーターで蒸発乾固した。 As shown in FIG. 1, it was found that the chart has two peaks on the front side and the back side, that is, the mixture is a mixture of two geometric isomers of cis form and trans form. Therefore, in order to identify the substance contained in each peak, the fractions of the front peak were collected and evaporated to dryness using a rotary evaporator.
（３）分取したピークの物質同定
前側ピークの蒸発乾固物と、cis-p-クマル酸とtrans-p-クマル酸の標品とをTLCで展開した。その結果を図２に示す。図２に示すように、標品との比較から、前側のピークがcis-p-クマル酸であることが分かった。反対に、後側のピークがtrans-p-クマル酸であることも分かった。
(3) Material identification of the collected peak The evaporated product of the front peak and cis-p-coumaric acid and trans-p-coumaric acid samples were developed by TLC. The result is shown in FIG. As shown in FIG. 2, it was found from the comparison with the standard that the front peak was cis-p-coumaric acid. Conversely, it was also found that the rear peak was trans-p-coumaric acid.
また、HPLCを使用して、前側ピークの蒸発乾固物と、cis-p-クマル酸とtrans-p-クマル酸の標品の保持時間（Retention time）を測定した。その結果を図３に示す。標品との比較から、前側のピークがcis-p-クマル酸に近い保持時間を有することが分かった。すなわち、前側のピークがcis-p-クマル酸であることが分かった。反対に、後側のピークがtrans-p-クマル酸であることも分かった。 Moreover, the retention time (Retention time) of the evaporative dry product of the front peak and the cis-p-coumaric acid and trans-p-coumaric acid samples were measured using HPLC. The result is shown in FIG. From the comparison with the sample, it was found that the front peak has a retention time close to that of cis-p-coumaric acid. That is, the front peak was found to be cis-p-coumaric acid. Conversely, it was also found that the rear peak was trans-p-coumaric acid.
さらに、前側ピークの蒸発乾固物とtrans-p-クマル酸の標品をNMRで測定した。その結果を図４に示す。標品との比較から、前側のピークがtrans-p-クマル酸とは異なる分子構造を有することが分かった。すなわち、前側のピークは、trans-p-クマル酸ではなく、cis-p-クマル酸であることが分かった。反対に、後側のピークがtrans-p-クマル酸であることも分かった。 Furthermore, the evaporative solid product of the front peak and a sample of trans-p-coumaric acid were measured by NMR. The result is shown in FIG. From comparison with the standard product, it was found that the front peak had a molecular structure different from trans-p-coumaric acid. That is, it was found that the front peak was not trans-p-coumaric acid but cis-p-coumaric acid. Conversely, it was also found that the rear peak was trans-p-coumaric acid.
以上のように、cis-p-クマル酸とtrans-p-クマル酸の混合物は、液体クロマトグラフィーによって高い分解能で前後２つのピークに分離でき、そのうちの前側ピークがcis-p-クマル酸に該当することがTLC、HPLC、NMRによって確認できた。 As described above, the mixture of cis-p-coumaric acid and trans-p-coumaric acid can be separated into two front and rear peaks with high resolution by liquid chromatography, of which the front peak corresponds to cis-p-coumaric acid This was confirmed by TLC, HPLC, and NMR.
３．他の桂皮酸類について
p-クマル酸と同様の方法で、カフェ酸及びフェルラ酸についても液体クロマトグラフィーで分離精製した。カフェ酸についての液体クロマトグラフィーのチャートを図５に示す。また、フェルラ酸についての液体クロマトグラフィーのチャートを図６に示す。
3. About other cinnamic acids
Caffeic acid and ferulic acid were also separated and purified by liquid chromatography in the same manner as p-coumaric acid. A liquid chromatography chart for caffeic acid is shown in FIG. Moreover, the chart of the liquid chromatography about ferulic acid is shown in FIG.
図５及び図６に示すように、p-クマル酸と同様に、カフェ酸及びフェルラ酸でもcis体とtrans体を示す２つのピークを確認できた。すなわち、本発明の分離精製法は、カフェ酸及びフェルラ酸でも有効であった。 As shown in FIGS. 5 and 6, two peaks indicating cis and trans isomers were confirmed in caffeic acid and ferulic acid as in p-coumaric acid. That is, the separation and purification method of the present invention was effective even with caffeic acid and ferulic acid.
分離精製した桂皮酸類のcis体のみを含む抗ウイルス剤と、trans体のみを含む抗ウイルス剤の効果を調べて比較した。以下に詳説する。 We investigated and compared the effects of the antiviral agent containing only the cis form of the cinnamic acid isolated and purified, and the antiviral agent containing only the trans form. The details are described below.
１．実験材料
（１）ウイルス
A型インフルエンザウイルスAichi株（IAV/Aichi）を使用した。
1. Experimental material (1) Virus
Influenza A virus strain Aichi (IAV / Aichi) was used.
（２）細胞及び細胞培養用培地
A型インフルエンザウイルスAichi株の感染価の測定には、イヌ腎由来のMDCK細胞を使用した。なお、細胞培養には、5%ウシ胎児血清（以下、FBSと省略する。) を含むイーグル最低必須培地（以下、MEMと省略する。）を使用した。
(2) Cells and cell culture medium
MDCK cells derived from canine kidney were used to measure the infectious titer of influenza A virus strain Aichi. For cell culture, Eagle's minimum essential medium (hereinafter abbreviated as MEM) containing 5% fetal bovine serum (hereinafter abbreviated as FBS) was used.
２．実験方法
（３）ウイルスの定量
ウイルスはプラーク法で定量した。具体的には以下のようにして定量した。まず、ウイルスの定量に使用する細胞が50mm‐ディッシュの底部全体を覆うようになるまで単層培養した。つぎに、ウイルス試料をリン酸緩衝生理食塩水（PBS）で10倍階段希釈し、その0.5mlをディッシュに接種したのち、室温で１時間ゆっくりと機械的に振盪して、ウイルスを吸着させた。なお、ウイルスの非特異的な不活化を抑えるために、希釈液には0.1％ウシ血清アルブミン（以下、BSAと省略する。）を加えた。
2. Experimental method (3) Quantification of virus Virus was quantified by the plaque method. Specifically, it was quantified as follows. First, monolayer culture was performed until the cells used for virus quantification covered the entire bottom of the 50 mm-dish. Next, the virus sample was serially diluted 10 times with phosphate buffered saline (PBS), 0.5 ml was inoculated into a dish, and then gently shaken mechanically at room temperature for 1 hour to adsorb the virus. . In order to suppress nonspecific inactivation of the virus, 0.1% bovine serum albumin (hereinafter abbreviated as BSA) was added to the diluted solution.
（４）A型インフルエンザウイルスAichi株（IAV/Aichi）に対する不活化
cis-p-クマル酸とtrans-p-クマル酸をエタノールに溶かし0.5 M溶液を調製した。この溶液を使用して、種々の濃度（5.0 mM, 7.5 mM, 10.0 mM）のcis-p-クマル酸又はtrans-p-クマル酸を含む10ｍMクエン酸緩衝液（pH 5.2, pH 5.5, pH 6.0)を調製した。緩衝液をプラスチック製のチューブ（Assist tube）に一定量ずつ加え氷冷した。
(4) Inactivation against influenza A virus strain Aichi (IAV / Aichi)
A 0.5 M solution was prepared by dissolving cis-p-coumaric acid and trans-p-coumaric acid in ethanol. Using this solution, 10 mM citrate buffer (pH 5.2, pH 5.5, pH 6.0) containing cis-p-coumaric acid or trans-p-coumaric acid at various concentrations (5.0 mM, 7.5 mM, 10.0 mM) ) Was prepared. A fixed amount of the buffer solution was added to a plastic tube (Assist tube) and cooled on ice.
各チューブ内容量の1/19量になるように、A型インフルエンザウイルスAichi株（IAV/Aichi）を含むウイルス液を添加し、30℃で10分間保温した。なお、クマル酸を含む10ｍMクエン酸緩衝液の代わりに、PBSに同じウイルス液を添加し、同様に保温して、実験対照とした。 A virus solution containing influenza A virus strain Aichi (IAV / Aichi) was added so that the volume in each tube was 1/19, and the mixture was kept at 30 ° C. for 10 minutes. In addition, instead of the 10 mM citrate buffer containing coumaric acid, the same virus solution was added to PBS and kept in the same manner as an experimental control.
保温後、BSAを加えた冷たいウイルス希釈液で直ちに各チューブ内の内容物を10倍階段希釈し、ウイルスの非特異的な不活化を抑えた。クマル酸を含むチューブの残存感染性ウイルス量と、実験対照のチューブの感染性ウイルス量とを測定した。両者の比を残存感染性ウイルス量とし、これをグラフにした。その結果を図７に示す。 After the incubation, the contents in each tube were immediately diluted 10-fold with a cold virus diluted solution containing BSA to suppress nonspecific inactivation of the virus. The amount of infectious virus remaining in the tube containing coumaric acid and the amount of infectious virus in the experimental control tube were measured. The ratio of the two was the amount of residual infectious virus, and this was graphed. The result is shown in FIG.
ここで、cis-p-クマル酸は白シンボル（○、△、□）、trans-p-クマル酸は黒シンボル（●、▲、■）で示している。また、処理溶液のpHに応じて四角、丸、三角のシンボルをpH 5.2 (□、■), pH 5.5 (○、●), pH 6.0 (△、▲)のように使用している。 Here, cis-p-coumaric acid is indicated by a white symbol (◯, Δ, □), and trans-p-coumaric acid is indicated by a black symbol (●, ▲, ■). In addition, square, circle, and triangle symbols are used as pH 5.2 (□, ■), pH 5.5 (◯, ●), pH 6.0 (△, ▲) depending on the pH of the treatment solution.
３．実験結果
図７から、cis-p-クマル酸及びtrans-p-クマル酸の存在下では、その濃度に依存したウイルス不活化が見られ、かつ、不活化は酸性側でより顕著であった。また、何れのpHにおいても、顕著な不活化が見られる条件下では、cis-p-クマル酸がtrans-p-クマル酸よりも明らかに強いウイルス不活化活性を示していた。
3. Experimental Results From FIG. 7, in the presence of cis-p-coumaric acid and trans-p-coumaric acid, virus inactivation depending on the concentration was observed, and the inactivation was more remarkable on the acidic side. Moreover, cis-p-coumaric acid showed a clearly stronger virus inactivating activity than trans-p-coumaric acid under conditions where remarkable inactivation was observed at any pH.
Claims (6)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2016171458A JP6802542B2 (en) | 2016-09-02 | 2016-09-02 | Separation and purification method for cinnamon acid geometric isomers |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2016171458A JP6802542B2 (en) | 2016-09-02 | 2016-09-02 | Separation and purification method for cinnamon acid geometric isomers |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2018036214A true JP2018036214A (en) | 2018-03-08 |
| JP6802542B2 JP6802542B2 (en) | 2020-12-16 |
Family
ID=61565624
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2016171458A Active JP6802542B2 (en) | 2016-09-02 | 2016-09-02 | Separation and purification method for cinnamon acid geometric isomers |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP6802542B2 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109085283A (en) * | 2018-08-30 | 2018-12-25 | 江西省药品检验检测研究院 | A kind of measuring method of Baizi Yangxin Pills content |
| CN109991328A (en) * | 2019-04-04 | 2019-07-09 | 西安医学院 | A kind of affine cudweed one surveys the quality evaluating method more commented |
| JP2020085588A (en) * | 2018-11-21 | 2020-06-04 | ＢＧＧ Ｊａｐａｎ株式会社 | Method for determining saw palmetto oil |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS60158119A (en) * | 1984-01-30 | 1985-08-19 | Daicel Chem Ind Ltd | Separating agent |
| JPH0585947A (en) * | 1991-09-25 | 1993-04-06 | Daicel Chem Ind Ltd | Chromatographic separation method |
| JP2005337946A (en) * | 2004-05-28 | 2005-12-08 | Kuraray Co Ltd | Method of separating geometrical isomer of 6,10,14-trimethyl-pentadaca-5,9,13-triene-2-on |
| US20150335689A1 (en) * | 2000-10-04 | 2015-11-26 | Paul Edward Stamets | Integrative fungal solutions for protecting bees |
| WO2016024359A1 (en) * | 2014-08-15 | 2016-02-18 | 株式会社カスケード資源研究所 | Pharmaceutical agent including lignin extract as active ingredient |
-
2016
- 2016-09-02 JP JP2016171458A patent/JP6802542B2/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS60158119A (en) * | 1984-01-30 | 1985-08-19 | Daicel Chem Ind Ltd | Separating agent |
| JPH0585947A (en) * | 1991-09-25 | 1993-04-06 | Daicel Chem Ind Ltd | Chromatographic separation method |
| US20150335689A1 (en) * | 2000-10-04 | 2015-11-26 | Paul Edward Stamets | Integrative fungal solutions for protecting bees |
| JP2005337946A (en) * | 2004-05-28 | 2005-12-08 | Kuraray Co Ltd | Method of separating geometrical isomer of 6,10,14-trimethyl-pentadaca-5,9,13-triene-2-on |
| WO2016024359A1 (en) * | 2014-08-15 | 2016-02-18 | 株式会社カスケード資源研究所 | Pharmaceutical agent including lignin extract as active ingredient |
Non-Patent Citations (5)
| Title |
|---|
| DARINKA BRODNJAK VONCINA ET AL.: "High-Performance Liquid Chromatographic Determination of Selected Phenolic Acids in Wine", NOVA BIOTECHNOLOGICA 9-2, JPN6020023918, 2009, pages 113 - 118, ISSN: 0004299945 * |
| FANG-MING SUN ET AL.: "Effect of Food Processing on Bioactive Compounds in Foods: A New Method foe Separation and Identific", AMERICAN CHEMICAL SOCIETY, JPN6020023914, 2002, pages 228 - 240, ISSN: 0004299944 * |
| KEHAN PEI ET AL.: "p-Coumaric acid and its conjugates:dietary sources, pharmacokinetic properties and biological activi", JOURNALSCIENCE FOOD AGRICALTURE, vol. 92巻, JPN6020023916, 2016, pages 2952 - 2962, ISSN: 0004299946 * |
| MING-JIE CHENN ET AL.: "Cis- and Trans-Cinnamic Acids Have Different Effects on the Catalytic Propertoes of Arabidopsis Phen", JOURNAL OF INTEGRATIVE PLANT BIOLOGY, vol. 47巻，1号, JPN6020023912, 2005, pages 67 - 75, ISSN: 0004299943 * |
| O.V. CARVALHO ET AL.: "In vitro inhibition of canine distemper virus by flavonoids and phenolic acids: Implications of stru", RESEARCH IN VETERINARY SCIENCE, vol. 95, JPN6020023919, 2013, pages 717 - 724, ISSN: 0004299947 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109085283A (en) * | 2018-08-30 | 2018-12-25 | 江西省药品检验检测研究院 | A kind of measuring method of Baizi Yangxin Pills content |
| CN109085283B (en) * | 2018-08-30 | 2020-07-10 | 江西省药品检验检测研究院 | Method for determining content of cedar seed heart nourishing pills |
| JP2020085588A (en) * | 2018-11-21 | 2020-06-04 | ＢＧＧ Ｊａｐａｎ株式会社 | Method for determining saw palmetto oil |
| JP7217134B2 (en) | 2018-11-21 | 2023-02-02 | ＢＧＧ Ｊａｐａｎ株式会社 | How to identify saw palmetto oil |
| CN109991328A (en) * | 2019-04-04 | 2019-07-09 | 西安医学院 | A kind of affine cudweed one surveys the quality evaluating method more commented |
| CN109991328B (en) * | 2019-04-04 | 2022-04-22 | 西安医学院 | Quality evaluation method for one-test-multiple evaluation of affine cudweed |
Also Published As
| Publication number | Publication date |
|---|---|
| JP6802542B2 (en) | 2020-12-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Mattioli et al. | Anthocyanins: A comprehensive review of their chemical properties and health effects on cardiovascular and neurodegenerative diseases | |
| Ichiyanagi et al. | Bioavailability and tissue distribution of anthocyanins in bilberry (Vaccinium myrtillus L.) extract in rats | |
| Ribeiro de Lima et al. | Determination of stilbenes (trans-astringin, cis-and trans-piceid, and cis-and trans-resveratrol) in Portuguese wines | |
| Careri et al. | Direct HPLC analysis of quercetin and trans-resveratrol in red wine, grape, and winemaking byproducts | |
| Wölwer-Rieck et al. | Investigations on the stability of stevioside and rebaudioside A in soft drinks | |
| Nayaka et al. | Cytoprotective and antioxidant activity studies of jaggery sugar | |
| Shia et al. | Metabolism and pharmacokinetics of 3, 3′, 4′, 7-tetrahydroxyflavone (fisetin), 5-hydroxyflavone, and 7-hydroxyflavone and antihemolysis effects of fisetin and its serum metabolites | |
| Zhao et al. | Unraveling the polypharmacology of a natural antifungal product, eugenol, against Rhizoctonia solani | |
| Watanabe et al. | Isolation and characterization of a phenolic antioxidant from the Pacific oyster (Crassostrea gigas) | |
| Burlec et al. | Phytochemical profile, antioxidant activity, and cytotoxicity assessment of Tagetes erecta L. flowers | |
| Tuenter et al. | Cyclopeptide alkaloids from Hymenocardia acida | |
| JP6802542B2 (en) | Separation and purification method for cinnamon acid geometric isomers | |
| WO2013061816A1 (en) | Novel compound contained in manuka honey and use of same | |
| Lu et al. | Oxyresveratrol, a stilbene compound from Morus alba L. twig extract active against Trichophyton rubrum | |
| Nakhate et al. | Therapeutic potential and pharmaceutical development of a multitargeted flavonoid phloretin | |
| AU2015391981A1 (en) | Plant-based biologically active substance having a polypharmacological effect | |
| Sekizawa et al. | Identification of the components in a vaccinium oldhamii extract showing inhibitory activity against influenza virus adsorption | |
| Li et al. | Study on separation and purification of genistein in the soybean residue using macroporous resin adsorption | |
| EP1292319A2 (en) | Extracts from spermatophyte plants with antitumor activity | |
| Lu et al. | Transformation of proanthocyanidin A2 to its isomers under different physiological pH conditions and common cell culture medium | |
| Destandau et al. | Gram-scale purification of dihydrorobinetin from Robinia pseudoacacia L. wood by centrifugal partition chromatography | |
| Yang et al. | Phenolic acid profiling of Lactarius hatsudake extracts, anti-cancer function and its molecular mechanisms | |
| Zhao et al. | The extraction and high antiproliferative effect of anthocyanin from gardenblue blueberry | |
| Matsui et al. | Three phlorotannins from Sargassum carpophyllum are effective against the secretion of allergic mediators from antigen-stimulated rat basophilic leukemia cells | |
| CN102267895B (en) | Phenylpropanoid compound as well as preparation method and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A80 | Written request to apply exceptions to lack of novelty of invention |
Free format text: JAPANESE INTERMEDIATE CODE: A80 Effective date: 20160928 |
|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20190809 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20190819 |
|
| A977 | Report on retrieval |
Free format text: JAPANESE INTERMEDIATE CODE: A971007 Effective date: 20200527 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20200707 |
|
| RD04 | Notification of resignation of power of attorney |
Free format text: JAPANESE INTERMEDIATE CODE: A7424 Effective date: 20200701 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20200901 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20201027 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20201117 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 6802542 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |