JP6836229B2 - Virus growth inhibitor - Google Patents
Virus growth inhibitor Download PDFInfo
- Publication number
- JP6836229B2 JP6836229B2 JP2016157619A JP2016157619A JP6836229B2 JP 6836229 B2 JP6836229 B2 JP 6836229B2 JP 2016157619 A JP2016157619 A JP 2016157619A JP 2016157619 A JP2016157619 A JP 2016157619A JP 6836229 B2 JP6836229 B2 JP 6836229B2
- Authority
- JP
- Japan
- Prior art keywords
- virus
- egcg
- growth inhibitor
- fraction
- proanthocyanidins
- 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.)
- Active
Links
- 241000700605 Viruses Species 0.000 title claims description 51
- 239000003966 growth inhibitor Substances 0.000 title claims description 28
- 230000002401 inhibitory effect Effects 0.000 claims description 26
- 229920002770 condensed tannin Polymers 0.000 claims description 25
- 239000004480 active ingredient Substances 0.000 claims description 12
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims description 12
- 235000013305 food Nutrition 0.000 claims description 8
- 239000003814 drug Substances 0.000 claims description 7
- 241000714201 Feline calicivirus Species 0.000 claims description 5
- 229940079593 drug Drugs 0.000 claims description 5
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 5
- 241000711573 Coronaviridae Species 0.000 claims description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 39
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 30
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 17
- 241000315672 SARS coronavirus Species 0.000 description 15
- 244000269722 Thea sinensis Species 0.000 description 15
- 238000000034 method Methods 0.000 description 15
- 229920001991 Proanthocyanidin Polymers 0.000 description 14
- JPFCOVZKLAXXOE-XBNSMERZSA-N (3r)-2-(3,5-dihydroxy-4-methoxyphenyl)-8-[(2r,3r,4r)-3,5,7-trihydroxy-2-(4-hydroxyphenyl)-3,4-dihydro-2h-chromen-4-yl]-3,4-dihydro-2h-chromene-3,5,7-triol Chemical compound C1=C(O)C(OC)=C(O)C=C1C1[C@H](O)CC(C(O)=CC(O)=C2[C@H]3C4=C(O)C=C(O)C=C4O[C@@H]([C@@H]3O)C=3C=CC(O)=CC=3)=C2O1 JPFCOVZKLAXXOE-XBNSMERZSA-N 0.000 description 13
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 12
- 235000006468 Thea sinensis Nutrition 0.000 description 12
- WMBWREPUVVBILR-UHFFFAOYSA-N GCG Natural products C=1C(O)=C(O)C(O)=CC=1C1OC2=CC(O)=CC(O)=C2CC1OC(=O)C1=CC(O)=C(O)C(O)=C1 WMBWREPUVVBILR-UHFFFAOYSA-N 0.000 description 11
- 150000001765 catechin Chemical class 0.000 description 10
- ADRVNXBAWSRFAJ-UHFFFAOYSA-N catechin Natural products OC1Cc2cc(O)cc(O)c2OC1c3ccc(O)c(O)c3 ADRVNXBAWSRFAJ-UHFFFAOYSA-N 0.000 description 10
- 235000005487 catechin Nutrition 0.000 description 10
- LNTHITQWFMADLM-UHFFFAOYSA-N gallic acid Chemical compound OC(=O)C1=CC(O)=C(O)C(O)=C1 LNTHITQWFMADLM-UHFFFAOYSA-N 0.000 description 10
- 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 9
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 9
- 235000019253 formic acid Nutrition 0.000 description 9
- 238000004519 manufacturing process Methods 0.000 description 9
- 230000003612 virological effect Effects 0.000 description 9
- 210000004027 cell Anatomy 0.000 description 8
- 239000002032 methanolic fraction Substances 0.000 description 8
- 239000000203 mixture Substances 0.000 description 8
- 238000000746 purification Methods 0.000 description 8
- 230000000694 effects Effects 0.000 description 7
- XMOCLSLCDHWDHP-IUODEOHRSA-N epi-Gallocatechin Chemical compound C1([C@H]2OC3=CC(O)=CC(O)=C3C[C@H]2O)=CC(O)=C(O)C(O)=C1 XMOCLSLCDHWDHP-IUODEOHRSA-N 0.000 description 7
- 238000002953 preparative HPLC Methods 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 235000013616 tea Nutrition 0.000 description 7
- 229910021642 ultra pure water Inorganic materials 0.000 description 7
- 239000012498 ultrapure water Substances 0.000 description 7
- 241000712461 unidentified influenza virus Species 0.000 description 7
- 238000004440 column chromatography Methods 0.000 description 6
- 239000000539 dimer Substances 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- 230000009385 viral infection Effects 0.000 description 6
- WMBWREPUVVBILR-WIYYLYMNSA-N (-)-Epigallocatechin-3-o-gallate Chemical compound O([C@@H]1CC2=C(O)C=C(C=C2O[C@@H]1C=1C=C(O)C(O)=C(O)C=1)O)C(=O)C1=CC(O)=C(O)C(O)=C1 WMBWREPUVVBILR-WIYYLYMNSA-N 0.000 description 5
- 235000020279 black tea Nutrition 0.000 description 5
- 235000020333 oolong tea Nutrition 0.000 description 5
- 239000002245 particle Substances 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 235000014347 soups Nutrition 0.000 description 5
- 238000003786 synthesis reaction Methods 0.000 description 5
- 210000003501 vero cell Anatomy 0.000 description 5
- 239000003054 catalyst Substances 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 239000000706 filtrate Substances 0.000 description 4
- 229940074391 gallic acid Drugs 0.000 description 4
- 235000004515 gallic acid Nutrition 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 238000005259 measurement Methods 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- LSHVYAFMTMFKBA-UHFFFAOYSA-N ECG Natural products C=1C=C(O)C(O)=CC=1C1OC2=CC(O)=CC(O)=C2CC1OC(=O)C1=CC(O)=C(O)C(O)=C1 LSHVYAFMTMFKBA-UHFFFAOYSA-N 0.000 description 3
- 241000282326 Felis catus Species 0.000 description 3
- 241000725303 Human immunodeficiency virus Species 0.000 description 3
- KDLHZDBZIXYQEI-UHFFFAOYSA-N Palladium Chemical compound [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 208000036142 Viral infection Diseases 0.000 description 3
- 240000006365 Vitis vinifera Species 0.000 description 3
- 235000014787 Vitis vinifera Nutrition 0.000 description 3
- 239000012298 atmosphere Substances 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 235000013399 edible fruits Nutrition 0.000 description 3
- 210000003734 kidney Anatomy 0.000 description 3
- 150000008442 polyphenolic compounds Chemical class 0.000 description 3
- 235000013824 polyphenols Nutrition 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000003826 tablet Substances 0.000 description 3
- 229960005486 vaccine Drugs 0.000 description 3
- PFTAWBLQPZVEMU-DZGCQCFKSA-N (+)-catechin Chemical compound C1([C@H]2OC3=CC(O)=CC(O)=C3C[C@@H]2O)=CC=C(O)C(O)=C1 PFTAWBLQPZVEMU-DZGCQCFKSA-N 0.000 description 2
- KTLUHRSHFRODPS-UHFFFAOYSA-N (+)-catechin-(4alpha,8)-(+)-catechin digallate Natural products C=1C=C(O)C(O)=CC=1C1OC2=CC(O)=CC(O)=C2C(C=2C=3OC(C(OC(=O)C=4C=C(O)C(O)=C(O)C=4)CC=3C(O)=CC=2O)C=2C=C(O)C(O)=CC=2)C1OC(=O)C1=CC(O)=C(O)C(O)=C1 KTLUHRSHFRODPS-UHFFFAOYSA-N 0.000 description 2
- PBVAJRFEEOIAGW-UHFFFAOYSA-N 3-[bis(2-carboxyethyl)phosphanyl]propanoic acid;hydrochloride Chemical compound Cl.OC(=O)CCP(CCC(O)=O)CCC(O)=O PBVAJRFEEOIAGW-UHFFFAOYSA-N 0.000 description 2
- 101100313763 Arabidopsis thaliana TIM22-2 gene Proteins 0.000 description 2
- 229920002330 B type proanthocyanidin Polymers 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 239000006145 Eagle's minimal essential medium Substances 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 241000709661 Enterovirus Species 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 241001149162 Mallotus japonicus Species 0.000 description 2
- 244000070406 Malus silvestris Species 0.000 description 2
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 244000299461 Theobroma cacao Species 0.000 description 2
- 235000021016 apples Nutrition 0.000 description 2
- 239000012223 aqueous fraction Substances 0.000 description 2
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- 239000000470 constituent Substances 0.000 description 2
- 239000002532 enzyme inhibitor Substances 0.000 description 2
- 239000012091 fetal bovine serum Substances 0.000 description 2
- 239000008187 granular material Substances 0.000 description 2
- 229940094952 green tea extract Drugs 0.000 description 2
- 235000020688 green tea extract Nutrition 0.000 description 2
- 235000015243 ice cream Nutrition 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 229920000609 methyl cellulose Polymers 0.000 description 2
- 239000001923 methylcellulose Substances 0.000 description 2
- 239000012454 non-polar solvent Substances 0.000 description 2
- 125000001997 phenyl group Chemical group [H]C1=C([H])C([H])=C(*)C([H])=C1[H] 0.000 description 2
- 239000002798 polar solvent Substances 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- VLFKNLZNDSEVBZ-UHFFFAOYSA-N procyanidin B-2 monogallate Natural products OC1C(C=2C=C(O)C(O)=CC=2)OC2=CC(O)=CC(O)=C2C1C(C=1OC2C=3C=C(O)C(O)=CC=3)=C(O)C=C(O)C=1CC2OC(=O)C1=CC(O)=C(O)C(O)=C1 VLFKNLZNDSEVBZ-UHFFFAOYSA-N 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 229910052938 sodium sulfate Inorganic materials 0.000 description 2
- 235000011152 sodium sulphate Nutrition 0.000 description 2
- 238000000638 solvent extraction Methods 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 241001529453 unidentified herpesvirus Species 0.000 description 2
- XMOCLSLCDHWDHP-SWLSCSKDSA-N (+)-Epigallocatechin Natural products C1([C@H]2OC3=CC(O)=CC(O)=C3C[C@@H]2O)=CC(O)=C(O)C(O)=C1 XMOCLSLCDHWDHP-SWLSCSKDSA-N 0.000 description 1
- 229930013915 (+)-catechin Natural products 0.000 description 1
- 235000007219 (+)-catechin Nutrition 0.000 description 1
- 229930013884 (+)-gallocatechin Natural products 0.000 description 1
- 235000007243 (+)-gallocatechin Nutrition 0.000 description 1
- 229930013783 (-)-epicatechin Natural products 0.000 description 1
- 235000007355 (-)-epicatechin Nutrition 0.000 description 1
- PFTAWBLQPZVEMU-UKRRQHHQSA-N (-)-epicatechin Chemical compound C1([C@H]2OC3=CC(O)=CC(O)=C3C[C@H]2O)=CC=C(O)C(O)=C1 PFTAWBLQPZVEMU-UKRRQHHQSA-N 0.000 description 1
- SYTBZMRGLBWNTM-SNVBAGLBSA-N (R)-flurbiprofen Chemical compound FC1=CC([C@H](C(O)=O)C)=CC=C1C1=CC=CC=C1 SYTBZMRGLBWNTM-SNVBAGLBSA-N 0.000 description 1
- KTLUHRSHFRODPS-RIQPQZJCSA-N 3,3'-Digalloylprocyanidin B2 Chemical compound O([C@@H]1[C@H](C2=C(O)C=C(C=C2O[C@@H]1C=1C=C(O)C(O)=CC=1)O)C=1C=2O[C@@H]([C@H](OC(=O)C=3C=C(O)C(O)=C(O)C=3)CC=2C(O)=CC=1O)C=1C=C(O)C(O)=CC=1)C(=O)C1=CC(O)=C(O)C(O)=C1 KTLUHRSHFRODPS-RIQPQZJCSA-N 0.000 description 1
- 235000017060 Arachis glabrata Nutrition 0.000 description 1
- 244000105624 Arachis hypogaea Species 0.000 description 1
- 235000010777 Arachis hypogaea Nutrition 0.000 description 1
- 235000018262 Arachis monticola Nutrition 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000282465 Canis Species 0.000 description 1
- 241000282552 Chlorocebus aethiops Species 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- 241000450599 DNA viruses Species 0.000 description 1
- 239000004278 EU approved seasoning Substances 0.000 description 1
- 241001289529 Fallopia multiflora Species 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 244000309467 Human Coronavirus Species 0.000 description 1
- 241000712431 Influenza A virus Species 0.000 description 1
- 241000713196 Influenza B virus Species 0.000 description 1
- 241000713297 Influenza C virus Species 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 229920000168 Microcrystalline cellulose Polymers 0.000 description 1
- 241000127282 Middle East respiratory syndrome-related coronavirus Species 0.000 description 1
- 244000294411 Mirabilis expansa Species 0.000 description 1
- 235000015429 Mirabilis expansa Nutrition 0.000 description 1
- 235000009134 Myrica cerifera Nutrition 0.000 description 1
- 244000132436 Myrica rubra Species 0.000 description 1
- 235000014631 Myrica rubra Nutrition 0.000 description 1
- 102000004316 Oxidoreductases Human genes 0.000 description 1
- 108090000854 Oxidoreductases Proteins 0.000 description 1
- 241001631646 Papillomaviridae Species 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 235000014443 Pyrus communis Nutrition 0.000 description 1
- 240000001341 Reynoutria japonica Species 0.000 description 1
- 235000018167 Reynoutria japonica Nutrition 0.000 description 1
- 241000219061 Rheum Species 0.000 description 1
- 241000700584 Simplexvirus Species 0.000 description 1
- 244000061457 Solanum nigrum Species 0.000 description 1
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 description 1
- 102000019197 Superoxide Dismutase Human genes 0.000 description 1
- 108010012715 Superoxide dismutase Proteins 0.000 description 1
- 235000005764 Theobroma cacao ssp. cacao Nutrition 0.000 description 1
- 235000005767 Theobroma cacao ssp. sphaerocarpum Nutrition 0.000 description 1
- 241000219094 Vitaceae Species 0.000 description 1
- 235000009754 Vitis X bourquina Nutrition 0.000 description 1
- 235000012333 Vitis X labruscana Nutrition 0.000 description 1
- TVXBFESIOXBWNM-UHFFFAOYSA-N Xylitol Natural products OCCC(O)C(O)C(O)CCO TVXBFESIOXBWNM-UHFFFAOYSA-N 0.000 description 1
- 239000000443 aerosol Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 235000013361 beverage Nutrition 0.000 description 1
- 235000008429 bread Nutrition 0.000 description 1
- 244000309464 bull Species 0.000 description 1
- 235000001046 cacaotero Nutrition 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 235000014171 carbonated beverage Nutrition 0.000 description 1
- 125000004403 catechin group Chemical group 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000007910 chewable tablet Substances 0.000 description 1
- 229940112822 chewing gum Drugs 0.000 description 1
- 235000015218 chewing gum Nutrition 0.000 description 1
- 235000019219 chocolate Nutrition 0.000 description 1
- QFLMUASKTWGRQE-JNIIMKSASA-N cinnamtannin A2 Chemical compound C1([C@@H]2[C@H](O)[C@H](C3=C(O)C=C(O)C=C3O2)C2=C3O[C@@H]([C@H](O)[C@H](C3=C(O)C=C2O)C2=C(O)C=C(O)C3=C2O[C@@H]([C@H](O)[C@H]3C=2C(O)=CC(O)=C3C[C@H]([C@H](OC3=2)C=2C=C(O)C(O)=CC=2)O)C=2C=C(O)C(O)=CC=2)C=2C=C(O)C(O)=CC=2)=CC=C(O)C(O)=C1 QFLMUASKTWGRQE-JNIIMKSASA-N 0.000 description 1
- 235000013353 coffee beverage Nutrition 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 239000006071 cream Substances 0.000 description 1
- 239000012043 crude product Substances 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 210000004748 cultured cell Anatomy 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 235000019543 dairy drink Nutrition 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 235000011850 desserts Nutrition 0.000 description 1
- 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 1
- 229940042406 direct acting antivirals neuraminidase inhibitors Drugs 0.000 description 1
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 description 1
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 241001493065 dsRNA viruses Species 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 239000002038 ethyl acetate fraction Substances 0.000 description 1
- 235000019225 fermented tea Nutrition 0.000 description 1
- 235000011194 food seasoning agent Nutrition 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 235000013376 functional food Nutrition 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 235000002532 grape seed extract Nutrition 0.000 description 1
- 235000021021 grapes Nutrition 0.000 description 1
- 235000009569 green tea Nutrition 0.000 description 1
- 230000009036 growth inhibition Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 208000037797 influenza A Diseases 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 235000015110 jellies Nutrition 0.000 description 1
- 239000008274 jelly Substances 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 239000010410 layer Substances 0.000 description 1
- 239000006210 lotion Substances 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 239000008268 mayonnaise Substances 0.000 description 1
- 235000010746 mayonnaise Nutrition 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- HEBKCHPVOIAQTA-UHFFFAOYSA-N meso ribitol Natural products OCC(O)C(O)C(O)CO HEBKCHPVOIAQTA-UHFFFAOYSA-N 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
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 235000013536 miso Nutrition 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 210000004400 mucous membrane Anatomy 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 description 1
- 239000002674 ointment Substances 0.000 description 1
- 235000020232 peanut Nutrition 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 239000002953 phosphate buffered saline Substances 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 235000015108 pies Nutrition 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 235000010204 pine bark Nutrition 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 230000000379 polymerizing effect Effects 0.000 description 1
- -1 proanthocyanidin gallate compound Chemical class 0.000 description 1
- VLFKNLZNDSEVBZ-BTWXELLASA-N procyanidin B2 3'-O-gallate Chemical compound O([C@@H]1CC=2C(O)=CC(O)=C(C=2O[C@@H]1C=1C=C(O)C(O)=CC=1)[C@H]1C2=C(O)C=C(O)C=C2O[C@@H]([C@@H]1O)C=1C=C(O)C(O)=CC=1)C(=O)C1=CC(O)=C(O)C(O)=C1 VLFKNLZNDSEVBZ-BTWXELLASA-N 0.000 description 1
- 235000011962 puddings Nutrition 0.000 description 1
- 239000012264 purified product Substances 0.000 description 1
- 239000003419 rna directed dna polymerase inhibitor Substances 0.000 description 1
- 239000002911 sialidase inhibitor Substances 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 235000014214 soft drink Nutrition 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 235000013322 soy milk Nutrition 0.000 description 1
- 235000011496 sports drink Nutrition 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 239000000454 talc Substances 0.000 description 1
- 229910052623 talc Inorganic materials 0.000 description 1
- 108010038851 tannase Proteins 0.000 description 1
- 239000003803 thymidine kinase inhibitor Substances 0.000 description 1
- 241000701161 unidentified adenovirus Species 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 239000000811 xylitol Substances 0.000 description 1
- 235000010447 xylitol Nutrition 0.000 description 1
- HEBKCHPVOIAQTA-SCDXWVJYSA-N xylitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)CO HEBKCHPVOIAQTA-SCDXWVJYSA-N 0.000 description 1
- 229960002675 xylitol Drugs 0.000 description 1
- 235000013618 yogurt Nutrition 0.000 description 1
Landscapes
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Description
本発明は、プロアントシアニジンガレート類やテアシネンシン類を有効成分とするウイルス増殖阻害剤に関する。 The present invention relates to a viral growth inhibitor containing proanthocyanidins and teasinensins as active ingredients.
ウイルス感染症の予防・治療には、抗原抗体反応を利用したワクチンの開発が主流であるが、ウイルスは変異速度が速いために新興ウイルスや再興ウイルスが現れ、ワクチンの開発が対応できていないのが現状である。また、ワクチン以外の手段としては、ヘルペスウイルスに対するチミジンキナーゼ阻害剤、ヒト免疫不全ウイルスに対する逆転写酵素阻害剤やプロテアーゼ阻害剤、インフルエンザウイルスに対するノイラミニダーゼ阻害剤などの酵素阻害剤が挙げられる。しかし、これら酵素阻害剤が適用できるウイルスは極めて限定的である。 For the prevention and treatment of viral infections, the development of vaccines using antigen-antibody reactions is the mainstream, but due to the high mutation rate of viruses, emerging viruses and re-emerging viruses have appeared, and vaccine development has not been supported. Is the current situation. Examples of means other than the vaccine include enzyme inhibitors such as thymidine kinase inhibitors against herpes virus, reverse transcriptase inhibitors and protease inhibitors against human immunodeficiency virus, and neuraminidase inhibitors against influenza virus. However, the viruses to which these enzyme inhibitors can be applied are extremely limited.
一方、植物由来成分であるポリフェノールは種々の生理活性を有し、ウイルスに対しては、ウイルスと直接に接触して細胞への吸着を防ぐことでその感染力を奪う不活化効果や、ウイルス感染した後のウイルスの増殖を阻害する効果が知られている。例えば、カテキン類の一種である(-)-エピガロカテキン-3-O-ガレートがインフルエンザウイルスの増殖を阻害すること(特許文献1)、リンゴやブドウ由来のプロアントシアニジン類(カテキン類の重合体)がSARSコロナウイルスの増殖を阻害すること(特許文献2)が報告されている。また、プロアントシアニジン類を含むピーナッツ種皮抽出物がヒト免疫不全ウイルスやSARSコロナウイルスの増殖を阻害すること(特許文献3)、エピカテキン四量体がSARSコロナウイルス、ネコカリシウイルス、インフルエンザウイルスの増殖を阻害すること(特許文献4)などが報告されている。しかし、プロアントシアニジン類に没食子酸が結合することで、種々のウイルスに対する増殖阻害活性が著しく向上することは何ら報告されていない。 On the other hand, polyphenol, which is a plant-derived component, has various physiological activities, and for viruses, it has an inactivating effect that deprives the infectivity by preventing adsorption to cells by directly contacting the virus, and virus infection. It is known to have the effect of inhibiting the growth of the virus after infection. For example, (-)-epigallocatechin-3-O-gallate, which is a kind of catechins, inhibits the growth of influenza virus (Patent Document 1), and proanthocyanidins derived from apples and grapes (polymers of catechins). ) Inhibits the growth of SARS coronavirus (Patent Document 2). In addition, peanut seed coat extract containing proanthocyanidins inhibits the growth of human immunodeficiency virus and SARS coronavirus (Patent Document 3), and epicatechin tetramer causes the growth of SARS coronavirus, cat calicivirus, and influenza virus. (Patent Document 4) and the like have been reported. However, it has not been reported that the binding of gallic acid to proanthocyanidins significantly improves the growth inhibitory activity against various viruses.
また、烏龍茶や紅茶に含まれるテアシネンシン類(プロアントシアニジン類とは異なるカテキン類の二量体)が単純ヘルペスウイルスを不活化すること(非特許文献1)が報告されているが、ウイルスの増殖を阻害する効果については全く報告されていない。 In addition, it has been reported that teasinencins (dimers of catechins different from proanthocyanidins) contained in oolong tea and black tea inactivate herpes simplex virus (Non-Patent Document 1), but the virus propagates. No inhibitory effect has been reported.
本発明の課題は、新興ウイルスや再興ウイルス感染症にも対抗し得る、種々のウイルスに対して効果を示す、より効果の高い新たなウイルス増殖阻害剤を提供することにある。 An object of the present invention is to provide a new virus growth inhibitor that is effective against various viruses and is highly effective against emerging viruses and re-emerging virus infections.
本発明者らは、上記課題を解決すべく鋭意研究を重ねた結果、驚くべきことに、プロアントシアニジン類に没食子酸が結合したプロアントシアニジンガレート類及びテアシネンシン類が高いウイルス増殖阻害活性を有することを見出し、本発明を完成するに至った。 As a result of intensive studies to solve the above problems, the present inventors have surprisingly found that proanthocyanidins and teasinencins in which gallic acid is bound to proanthocyanidins have high viral growth inhibitory activity. We have found and completed the present invention.
すなわち、本発明は、式(I)で表わされるプロアントシアニジンガレート類、及び式(II)で表わされるテアシネンシン類から選ばれる1種又は2種以上を有効成分として含有するウイルス増殖阻害剤を提供するものである。また、該ウイルス増殖阻害剤を含む、ウイルスの増殖を阻害又は減少させるための飲食品、医薬品や医薬部外品を提供するものである。 That is, the present invention provides a virus growth inhibitor containing one or more selected from the proanthocyanidins represented by the formula (I) and the teasinencins represented by the formula (II) as active ingredients. It is a thing. It also provides foods and drinks, pharmaceuticals and quasi-drugs for inhibiting or reducing virus growth, including the virus growth inhibitor.
本発明のウイルス増殖阻害剤は種々のウイルスに対して高い増殖阻害活性を有し、かつ、茶由来のポリフェノールに含まれる成分のため安全性が高い。また、該ウイルス増殖阻害剤はカテキン類二量体と低分子のため、他のプロアントシアニジン類と比較して合成が容易である。また、本発明のウイルス増殖阻害剤は、医薬品、サプリメント、飲食品などにも含有させることができ、これらを投与又は摂取することにより、ウイルスの増殖を抑制し、ウイルス感染症の予防又は軽減が期待できる。 The virus growth inhibitor of the present invention has high growth inhibitory activity against various viruses and is highly safe because it is a component contained in tea-derived polyphenols. In addition, since the virus growth inhibitor is a catechin dimer and a small molecule, it is easier to synthesize than other proanthocyanidins. In addition, the virus growth inhibitor of the present invention can be contained in pharmaceuticals, supplements, foods and drinks, etc., and by administering or ingesting these, virus growth can be suppressed and virus infection can be prevented or alleviated. You can expect it.
以下において本発明を詳細に説明する。
本発明のウイルス増殖阻害剤の有効成分である、式(I)で表わされるプロアントシアニジンガレート類は、プロアントシアニジン類の一種である。
The proanthocyanidin gallates represented by the formula (I), which are the active ingredients of the virus growth inhibitor of the present invention, are a kind of proanthocyanidins.
プロアントシアニジン類は、(+)-カテキン[(+)-C]、(+)-ガロカテキン[(+)-GC]、(-)-エピカテキン[(-)-EC]、(-)-エピガロカテキン[(-)-EGC]、(-)-エピカテキン-3-O-ガレート[(-)-ECg]、(-)-エピガロカテキン-3-O-ガレート[(-)-EGCg]などのカテキン類を構成ユニットとして、それらが複数個結合した重合ポリフェノールとして知られている。その結合様式の違いから、プロアントシアニジン類は、A型(構成ユニット間に4→8及び2→O→7、又は4→6及び2→O→7の結合を有する)と、B型(構成ユニット間に4→8又は4→6の結合を有する)とに分類される。B型プロアントシアニジン類は茶、ブドウ種子、リンゴ、松樹皮、カカオなど多くの植物に含まれることが知られており、茶生葉、緑茶、烏龍茶、紅茶からは以下の式(III)〜(V)で表わされる二量体のB型プロアントシアニジン類(R1乃至R4の具体例は、式の下方に列挙する)が単離されている。 Proanthocyanidins are (+)-catechin [(+)-C], (+)-gallocatechin [(+)-GC], (-)-epicatechin [(-)-EC], (-)-epi Galocatechin [(-)-EGC], (-)-Epigallocatechin-3-O-gallate [(-)-ECg], (-)-Epigallocatechin-3-O-gallate [(-)-EGCg] It is known as a polymerized polyphenol in which a plurality of catechins such as catechins are bound as a constituent unit. Due to the difference in the binding mode, proanthocyanidins are type A (having 4 → 8 and 2 → O → 7 or 4 → 6 and 2 → O → 7 binding between the constituent units) and type B (composition). (Has a 4 → 8 or 4 → 6 bond between units). B-type proanthocyanidins are known to be contained in many plants such as tea, grape seeds, apples, pine bark, and cacao. From tea leaves, green tea, oolong tea, and black tea, the following formulas (III) to (V) ) B-type proanthocyanidins ( specific examples of R 1 to R 4 are listed below the formula) have been isolated.
本発明のウイルス増殖阻害剤は、二量体のB型プロアントシアニジン類に没食子酸が結合した式(I)で表わされるプロアントシアニジンガレート類を有効成分とする。式(I)で表わされるプロアントシアニジンガレート類は、ウイルス増殖阻害活性が高いという観点から下記式(VI)で表されるプロアントシアニジンガレート類が好ましい。式(VI)で表わされるプロアントシアニジンガレート類の具体例としては、(-)-EC-(4β→8)-(-)-ECg(プロシアニジンB2-3’-O-ガレート)、(-)-ECg-(4β→8)-(-)-ECg(プロシアニジンB2-3,3’-ジ-O-ガレート)、(-)-EGC-(4β→8)-(-)-ECg、(-)-EGCg-(4β→8)-(-)-ECg、(-)-EGC-(4β→8)-(-)-EGCg(プロデルフィニジンB2-3’-O-ガレート)、(-)-EGCg-(4β→8)-(-)-EGCg(プロデルフィニジンB2-3,3’-ジ-O-ガレート)、(-)-EC-(4β→8)-(-)-EGCg、(+)-GC-(4α→8)-(-)-EGCg(プロデルフィニジンB4-3’-O-ガレート)、(+)-C-(4α→8)-(-)-EGCg、(+)-GC-(4α→8)-(-)-ECgなどが挙げられる。プロアントシアニジンガレート類は公知の化学的合成方法(例えば、Molecules, 20, 18870-18885 (2015)に記載されている方法)などにより製造することができる。具体例を挙げると、(-)-EGC-(4β→8)-(-)-EGCgは、ベンジル基で保護した(-)-EGCへ脱離基を導入し、ベンジル基で保護した(-)-EGCgと縮合させ、そして脱保護させることにより製造することが可能である。 The viral growth inhibitor of the present invention contains proanthocyanidin gallates represented by the formula (I) in which gallic acid is bound to dimers of type B proanthocyanidins as an active ingredient. The proanthocyanidin gallate represented by the formula (I) is preferably the proanthocyanidin gallate represented by the following formula (VI) from the viewpoint of high virus growth inhibitory activity. Specific examples of proanthocyanidin gallates represented by the formula (VI) include (-)-EC- (4β → 8)-(-)-ECg (procyanidin B2-3'-O-gallate), (-)-. ECg-(4β → 8)-(-)-ECg (procyanidin B2-3,3'-di-O-gallate), (-)-EGC-(4β → 8)-(-)-ECg, (-) -EGCg- (4β → 8)-(-)-ECg, (-)-EGC-(4β → 8)-(-)-EGCg (proanthocyanidin B2-3'-O-gallate), (-)-EGCg -(4β → 8)-(-)-EGCg (proanthocyanidin B2-3,3'-di-O-gallate), (-)-EC-(4β → 8)-(-)-EGCg, (+) -GC- (4α → 8)-(-)-EGCg (proanthocyanidin B4-3'-O-gallate), (+)-C-(4α → 8)-(-)-EGCg, (+)-GC -(4α → 8)-(-)-ECg and so on. Proanthocyanidins gallates can be produced by a known chemical synthesis method (for example, the method described in Molecules, 20, 18870-18885 (2015)). To give a specific example, (-)-EGC- (4β → 8)-(-)-EGCg introduced a leaving group into (-)-EGC protected with a benzyl group and protected with a benzyl group (-). )-Can be produced by condensing with EGCg and deprotecting.
また、本発明のウイルス増殖阻害剤の有効成分であるプロアントシアニジンガレート類は、茶(Camellia sinensis)の他、大黄(Rheum sp.)、ヤマモモ(Morella rubra)、ヨーロッパブドウ(Vitis vinifera)、ツルドクダミ(Polygonum multiflorum)、アカメガシワ(Mallotus japonicus)などを抽出・精製して得ることもできる。医薬上又は食品上許容し得る規格に適合し、本発明の効果を発揮するものであれば、プロアントシアニジンガレート類の粗精製物を用いてもよく、公知の分離精製方法を適宜組み合わせて純度を上げたものを用いてもよい。精製手段としては、例えば水、熱水、アルコールなどの極性溶媒、又は非極性溶媒を用いて行う溶剤抽出法、遠心分離法、高速液体クロマトグラフ法やカラムクロマトグラフ法などが挙げられる。 In addition to tea (Camellia sinensis), proanthocyanidin gallates, which are the active ingredients of the virus growth inhibitor of the present invention, include tea (Camellia sinensis), mallotus japonicus (Rheum sp.), Bayberry (Morella rubra), European grape (Vitis vinifera), and tuber fleeceflower (Turdokudami). Polygonum multiflorum), Mallotus japonicus, etc. can also be extracted and purified. A crude product of proanthocyanidin gallates may be used as long as it conforms to pharmaceutically or food-acceptable standards and exhibits the effects of the present invention, and the purity is adjusted by appropriately combining known separation and purification methods. You may use the raised one. Examples of the purification means include a solvent extraction method using a polar solvent such as water, hot water, alcohol, or a non-polar solvent, a centrifugation method, a high-speed liquid chromatograph method, a column chromatograph method, and the like.
本発明のウイルス増殖阻害剤は、式(II)で表わされるテアシネンシン類を有効成分として含有することもできる。 The virus growth inhibitor of the present invention can also contain teasinensins represented by the formula (II) as an active ingredient.
上記式には、説明のため所定のベンゼン環にBの文字を付し、そのベンゼン環がB環であることを示している。
テアシネンシン類は、カテキン類がB環同士で結合した二量体であり、烏龍茶や紅茶に含まれる。良く知られたテアシネンシン類としては以下の式(VII)や(VIII)で表わされるテアシネンシンA、テアシネンシンB、テアシネンシンC、テアシネンシンD、テアシネンシンEが挙げられる。
In the above formula, a letter B is added to a predetermined benzene ring for explanation, and it is shown that the benzene ring is a B ring.
Teacinencins are dimers in which catechins are bound to each other in the B ring, and are contained in oolong tea and black tea. Well-known teasinencins include teasinencin A, teasinencin B, teasinencin C, teasinencin D, and teasinencin E represented by the following formulas (VII) and (VIII).
上記式中の結合態様を示す「S」、「R」は、それぞれS体、R体をそれぞれ表す。 "S" and "R" indicating the binding mode in the above formula represent S-form and R-form, respectively.
ウイルス増殖阻害活性が高いという観点から、下記式(IX)で表わされるテアシネンシンガレート類が好ましい。式(IX)で表わされるテアシネンシンガレート類としては、テアシネンシンA、テアシネンシンB、テアシネンシンDなどが例示できる。テアシネンシン類は公知の化学的合成法(例えば、特開2010-138103号公報、Chem. Pharm. Bull. 59, 1183-1185 (2011)等に記載の方法)やナシ果実ホモジネートを用いた方法(Tetrahedron, 59, 7939-7947 (2003)に記載の方法)などにより製造することができる。例えばテアシネンシンAは、触媒や酸化酵素と共に(-)-EGCgを酸化重合した後、還元して製造することができる。 From the viewpoint of high virus growth inhibitory activity, teasinen singalates represented by the following formula (IX) are preferable. Examples of the teasinencin gallates represented by the formula (IX) include teasinencin A, teasinencin B, and teasinencin D. For tetrahedrons, a known chemical synthesis method (for example, the method described in JP-A-2010-138103, Chem. Pharm. Bull. 59, 1183-1185 (2011), etc.) or a method using pear fruit homogenate (Tetrahedron) , 59, 7939-7947 (2003)) and the like. For example, teacinencin A can be produced by oxidatively polymerizing (-)-EGCg together with a catalyst or an oxidase and then reducing it.
また、本発明のウイルス増殖阻害剤の有効成分であるテアシネンシン類は、烏龍茶などの半発酵茶や紅茶などの発酵茶を抽出・精製して得ることもできる。医薬上又は食品上許容し得る規格に適合し、本発明の効果を発揮するものであれば、テアシネンシン類の粗精製物を用いてもよく、公知の分離精製方法を適宜組み合わせて純度を上げたものを用いてもよい。精製手段としては、例えば水、熱水、アルコールなどの極性溶媒、又は非極性溶媒を用いて行う溶剤抽出法、遠心分離法、高速液体クロマトグラフ法やカラムクロマトグラフ法などが挙げられる。 Further, the teasinencins, which are the active ingredients of the viral growth inhibitor of the present invention, can also be obtained by extracting and purifying semi-fermented tea such as oolong tea and fermented tea such as black tea. A crudely purified product of teasinencins may be used as long as it conforms to pharmaceutically or food-acceptable standards and exhibits the effects of the present invention, and the purity has been increased by appropriately combining known separation and purification methods. You may use the thing. Examples of the purification means include a solvent extraction method using a polar solvent such as water, hot water, alcohol, or a non-polar solvent, a centrifugation method, a high-speed liquid chromatograph method, a column chromatograph method, and the like.
本発明のウイルス増殖阻害剤が有効なウイルスとしては、コロナウイルス(SARSコロナウイルス、MERSコロナウイルス、ヒトコロナウイルスなど)、カリシウイルス(ネコカリシウイルス、ノーウォークウイルスなど)、インフルエンザウイルス(A型インフルエンザウイルス、B型インフルエンザウイルス、C型インフルエンザウイルスなど)、ヒト免疫不全ウイルス、ヘルペスウイルス、アデノウイルス、パピローマウイルス、ライノウイルス、エンテロウイルスなどのRNAウイルスやDNAウイルスが例示できる。 Examples of viruses for which the virus growth inhibitor of the present invention is effective include coronavirus (SARS coronavirus, MERS coronavirus, human coronavirus, etc.), calicivirus (cat calicivirus, nowalk virus, etc.), and influenza virus (influenza A). Examples include RNA viruses and DNA viruses such as viruses, influenza B virus, influenza C virus, etc.), human immunodeficiency virus, herpes virus, adenovirus, papillomavirus, rhinovirus, enterovirus, etc.
本発明のウイルス増殖阻害剤が効果を示すメカニズムは不明であるが、種々のウイルスに効果を示すことからウイルスに共通の増殖機能に作用していると考えられる。プロアントシアニジン類で細胞を処理するとマンガンスーパーオキシドジスムターゼが誘導され、ウイルス増殖に要求されるスーパーオキシドの産生を抑制することで、ウイルスの増殖が阻害されると考えられている(Biochem. Biophys. Res. Commun., 261, 139-143(1999)、特許文献3参照)。本発明のウイルス増殖阻害剤も同様のメカニズムでウイルスの増殖を阻害していると考えられる。 The mechanism by which the virus growth inhibitor of the present invention is effective is unknown, but since it is effective against various viruses, it is considered that it acts on the growth function common to viruses. Treatment of cells with proanthocyanidins induces manganese superoxide dismutase, which is thought to inhibit viral growth by suppressing the production of superoxide, which is required for viral growth (Biochem. Biophys. Res). . Commun., 261, 139-143 (1999), see Patent Document 3). It is considered that the virus growth inhibitor of the present invention also inhibits virus growth by the same mechanism.
本発明のウイルス増殖阻害剤の有効成分であるプロアントシアニジンガレート類、及びテアシネンシン類は、細胞内でのウイルス増殖を阻害する作用を有するため、これらの有効成分を含有するウイルス増殖阻害剤を経口的あるいは非経口的に、ヒトを含む哺乳動物に投与し、ウイルス感染症の予防・治療、ウイルス産生抑制が期待できる。ウイルス増殖阻害剤を経口的に投与する場合の剤形としては、錠剤、顆粒剤、細粒剤、丸剤、散剤、カプセル剤、トローチ剤、チュアブル剤、液剤(ドリンク剤)などが挙げられる。また、該有効成分に薬学的に許容される担体を添加して外用の製剤とすることもできる。製剤の形態としては、液剤、ローション剤、軟膏剤、クリーム剤、ゲル剤、テープ剤、パッチ剤、エアゾール剤、スプレー剤、乳液剤などが挙げられ、皮膚表面や粘膜などから作用するものであれば、どのような形態でもよい。 Since proanthocyanidin gallates and teasinencins, which are the active ingredients of the virus growth inhibitor of the present invention, have an action of inhibiting virus growth in cells, a virus growth inhibitor containing these active ingredients is orally used. Alternatively, it can be administered parenterally to mammals including humans to prevent / treat viral infections and suppress viral production. Dosage forms for oral administration of viral growth inhibitors include tablets, granules, fine granules, pills, powders, capsules, troches, chewables, liquids (drinks) and the like. Further, a pharmaceutically acceptable carrier may be added to the active ingredient to prepare a preparation for external use. Examples of the form of the preparation include liquids, lotions, ointments, creams, gels, tapes, patches, aerosols, sprays, emulsions, etc., which act on the skin surface or mucous membranes. Any form may be used.
また、本発明のウイルス増殖阻害剤を飲食品に含有して摂取することもできる。飲食品としては特に制限はされないが、具体的に対象となる飲食品としては、茶系飲料、コーヒー飲料、炭酸飲料、果実飲料、果実酒類、野菜飲料、清涼飲料、乳飲料、乳酸菌飲料、ドリンク剤、スポーツドリンク、豆乳などの飲料類;アイスクリーム、アイスミルク、ラクトアイス、氷菓、ヨーグルト、プリン、ゼリーなどのデザート類;饅頭、羊羹、キャラメル、キャンディー、錠菓、スナック、クラッカー、ビスケット、クッキー、パイ、チョコレート、チューインガムなどの菓子類;和風スープ、洋風スープ、中華スープ、味噌汁などのスープ類;パン類;ジャム類;マヨネーズ、ドレッシングなどの調味料類;レトルトカレーなどのレトルト食品などを挙げることができる。さらに、本発明のウイルス増殖阻害剤を少なくとも一種以上を飲食品に含ませることによってその食品を、ウイルス増殖を阻害する効果を目的とした機能性食品にすることができる。 In addition, the virus growth inhibitor of the present invention can be contained in foods and drinks and ingested. The foods and drinks are not particularly limited, but the specific target foods and drinks are tea-based drinks, coffee drinks, carbonated drinks, fruit drinks, fruit liquors, vegetable drinks, soft drinks, dairy drinks, lactic acid bacteria drinks, and drinks. Beverages such as medicines, sports drinks and soy milk; desserts such as ice cream, ice milk, lacto ice, ice cream, yogurt, pudding and jelly; Confectionery such as pie, chocolate, chewing gum; Japanese-style soup, Western-style soup, Chinese soup, miso soup and other soups; Breads; Jams; Mayonnaise, dressing and other seasonings; Can be done. Furthermore, by including at least one or more of the virus growth inhibitor of the present invention in foods and drinks, the food can be made into a functional food for the purpose of inhibiting virus growth.
以下に実施例を挙げ、本発明をさらに詳しく説明する。ただし、本発明はこれに限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to this.
[製造例1]プロアントシアニジンガレート類の調製
超純水1.2Lに溶解したポリフェノンG(緑茶抽出物粉末、三井農林株式会社製)100gをDIAION HP20カラム(70mm×310mm、三菱化学株式会社製)に供し、超純水1.8L、20%、40%、60%、80%メタノール(水/メタノール=8/2、6/4、4/6、2/8(体積比))水溶液各3L、アセトン3Lで順次溶出した。各溶出液を減圧濃縮、凍結乾燥を行い、水画分46.02g、20%メタノール画分16.97g、40%メタノール画分20.28g、60%メタノール画分14.25g、80%メタノール画分1.87g、アセトン画分0.50gを得た。
[Production Example 1] Preparation of proanthocyanidins Galates 100 g of polyphenone G (green tea extract powder, manufactured by Mitsui Norin Co., Ltd.) dissolved in 1.2 L of ultrapure water is placed on a DIAION HP20 column (70 mm x 310 mm, manufactured by Mitsubishi Chemical Corporation). Ultrapure water 1.8L, 20%, 40%, 60%, 80% methanol (water / methanol = 8/2, 6/4, 4/6, 2/8 (volume ratio)) aqueous solution 3L each, acetone It was eluted sequentially with 3 L. Each eluate was concentrated under reduced pressure and lyophilized to produce a water fraction of 46.02 g, a 20% methanol fraction of 16.97 g, a 40% methanol fraction of 20.28 g, a 60% methanol fraction of 14.25 g, and an 80% methanol fraction of 1.87 g. An acetone fraction of 0.50 g was obtained.
メタノール75mLに溶解した40%メタノール画分19.70gをTOYOPEARL HW-40Cカラム(40mm×440mm、東ソー株式会社製)にて分画した(移動相:メタノール、流速14.7mL/min)。溶出液を濃縮、凍結乾燥して、画分1(保持時間0〜60分)8.04g、画分2(同60〜150分)10.72g、画分3(同150〜270分)0.53gを得た。 19.70 g of a 40% methanol fraction dissolved in 75 mL of methanol was fractionated with a TOYO PEARL HW-40C column (40 mm × 440 mm, manufactured by Tosoh Corporation) (mobile phase: methanol, flow rate 14.7 mL / min). Concentrate the eluate, freeze-dry it, and add 8.04 g of fraction 1 (holding time 0 to 60 minutes), 10.72 g of fraction 2 (60 to 150 minutes), and 0.53 g of fraction 3 (150 to 270 minutes). Obtained.
0.52gの画分3を、Mightysil RP-18 GP II(20×250mm、粒子径5μm、関東化学株式会社製)を用いた分取HPLCにより分画した(カラム温度:26℃、流速:16mL/min、移動相A:水/ギ酸=1000/2(体積比)、移動相B:メタノール、グラジエントプログラム:0〜50分まで移動相Bを体積比で10〜30%に上昇→50〜51分まで移動相Bを体積比で30〜95%に上昇→51〜60分まで移動相Bを体積比で95%に保持)。溶出液を濃縮、凍結乾燥して、画分3-1(保持時間0〜12分)3mg、画分3-2(同12〜15分)64mg、画分3-3(同15〜19分)48mg、画分3-4(同19〜22分)21mg、画分3-5(同22〜27分)31mg、画分3-6(同27〜31分)35mg、画分3-7(同31〜54分)142mg、画分3-8(同54〜58分)14mgを得た。 Fraction 3 of 0.52 g was fractionated by preparative HPLC using Mightysil RP-18 GP II (20 × 250 mm, particle size 5 μm, manufactured by Kanto Chemical Co., Inc.) (column temperature: 26 ° C., flow velocity: 16 mL / min, mobile phase A: water / formic acid = 1000/2 (volume ratio), mobile phase B: methanol, gradient program: 0 to 50 minutes mobile phase B increased to 10 to 30% by volume → 50 to 51 minutes Mobile phase B increased to 30-95% by volume → Mobile phase B was maintained at 95% by volume from 51 to 60 minutes). Concentrate and lyophilize the eluate, fraction 3-1 (holding time 0-12 minutes) 3 mg, fraction 3-2 (12-15 minutes) 64 mg, fraction 3-3 (15-19 minutes) ) 48 mg, fraction 3-4 (19-22 minutes) 21 mg, fraction 3-5 (22-27 minutes) 31 mg, fraction 3-6 (27-31 minutes) 35 mg, fraction 3-7 (31-54 minutes) 142 mg and fraction 3-8 (54-58 minutes) 14 mg were obtained.
63mgの画分3-2をMightysil RP-18 GP II(20×250mm、粒子径5μm)を用いた分取HPLCにより精製(移動相:水/酢酸エチル/ギ酸=970/30/1(体積比)、カラム温度:26℃、流速:16mL/min)を行い、(-)-EGC-(4β→8)-(-)-EGCgを2mgと、(+)-GC-(4α→8)-(-)-EGCgを37mg得た。 63 mg fraction 3-2 was purified by preparative HPLC using Mightysil RP-18 GP II (20 x 250 mm, particle size 5 μm) (mobile phase: water / ethyl acetate / formic acid = 970/30/1 (volume ratio) ), Column temperature: 26 ° C, flow velocity: 16 mL / min), (-)-EGC- (4β → 8)-(-)-EGCg was added to 2 mg, and (+)-GC- (4α → 8)- 37 mg of (-)-EGCg was obtained.
46mgの画分3-3をMightysil RP-18 GP II(20×250mm、粒子径5μm)を用いた分取HPLCにより2回の精製(1回目 移動相:水/酢酸エチル/ギ酸=920/80/1(体積比)、カラム温度:26℃、流速:16mL/min。2回目 移動相:水/アセトニトリル/ギ酸=920/80/1(体積比)、カラム温度:26℃、流速:16mL/min)を行い、(+)-GC-(4α→8)-(-)-ECgを16mg得た。 Purification of 46 mg fraction 3-3 by preparative HPLC using Mightysil RP-18 GP II (20 × 250 mm, particle size 5 μm) (1st mobile phase: water / ethyl acetate / formic acid = 920/80) / 1 (volume ratio), column temperature: 26 ° C, flow rate: 16 mL / min. Second mobile phase: water / acetonitrile / formic acid = 920/80/1 (volume ratio), column temperature: 26 ° C, flow rate: 16 mL / min min) was performed to obtain 16 mg of (+)-GC- (4α → 8)-(-)-ECg.
19mgの画分3-4をMightysil RP-18 GP II(20×250mm、粒子径5μm)を用いた分取HPLCにより精製(移動相:水/アセトニトリル/ギ酸=910/90/2(体積比)、カラム温度:26℃、流速:16mL/min)を行い、(-)-EGCg-(4β→8)-(-)-EGCgを11mg得た。 Purification of 19 mg fraction 3-4 by preparative HPLC using Mightysil RP-18 GP II (20 x 250 mm, particle size 5 μm) (mobile phase: water / acetonitrile / formic acid = 910/90/2 (volume ratio)) , Column temperature: 26 ° C., flow velocity: 16 mL / min) to obtain 11 mg of (-)-EGCg- (4β → 8)-(-)-EGCg.
[製造例2](-)-EGC-(4β→8)-(-)-EGCの調製
8mgの(-)-EGCg-(4β→8)-(-)-EGCgを超純水1.5mLに溶解し、スミチームTAN(タンナーゼ、新日本化学工業株式会社製)2mgを加え37℃で30分間反応させた。その後、Mightysil RP-18 GP II(20×250mm、粒子径5μm)を用いた分取HPLCにより反応液の精製(移動相:水/メタノール/ギ酸=950/50/1(体積比)、カラム温度:26℃、流速:16mL/min)を行い、(-)-EGC-(4β→8)-(-)-EGCを5mg得た。
[Production Example 2] Preparation of (-)-EGC- (4β → 8)-(-)-EGC
Dissolve 8 mg of (-)-EGCg- (4β → 8)-(-)-EGCg in 1.5 mL of ultrapure water, add 2 mg of Sumiteam TAN (Tannase, manufactured by Shin Nihon Kagaku Kogyo Co., Ltd.), and add 2 mg at 37 ° C for 30 minutes. It was reacted. Then, the reaction solution was purified by preparative HPLC using Mightysil RP-18 GP II (20 x 250 mm, particle size 5 μm) (mobile phase: water / methanol / formic acid = 950/50/1 (volume ratio), column temperature. : 26 ° C., flow velocity: 16 mL / min) to obtain 5 mg of (-)-EGC- (4β → 8)-(-)-EGC.
[製造例3]テアシネンシンAの合成
1.8gの(-)-EGCg(三井農林株式会社製)を超純水60mLとMcIlvaine緩衝液(pH 5)60mLで溶解した。50mLコニカルビーカー4つそれぞれにこの溶液30mLと10%パラジウム/炭素(50%水湿潤品、SIGMA-ALDRICH社製)170mgを加えて、空気雰囲気下室温で180分間撹拌した。触媒をろ別した後、ろ液にジチオスレイトール463mgを加えて、室温で45分間撹拌した。反応液に1M水酸化ナトリウム水溶液を加えてpH6に調整した。反応液を酢酸エチル50mLで5回抽出し、酢酸エチル層を硫酸ナトリウムで脱水した。硫酸ナトリウムをろ別後、ろ液をロータリーエバポレーターで濃縮乾固した。酢酸エチル画分1.5gをメタノール5mLで溶解後、TOYOPEARL HW-40Sカラムクロマトグラフィー(40mm×450mm、移動相:メタノール、流速:15mL/min)に供し、粗テアシネンシンAを374mg得た。得られた粗テアシネンシンAを5%アセトニトリル(水/アセトニトリル=9.5/5、体積比)15mLに溶解後、MCIGEL-CHP55Yカラムクロマトグラフィー(20mm×300mm、三菱化学株式会社製、移動相:水/アセトニトリル/ギ酸=870/130/1(体積比)、流速:7.5mL/min)により精製し、テアシネンシンAを286mg得た。
[Production Example 3] Synthesis of teasinencin A
1.8 g of (-)-EGCg (manufactured by Mitsui Norin Co., Ltd.) was dissolved in 60 mL of ultrapure water and 60 mL of McIlvaine buffer (pH 5). To each of the four 50 mL conical beakers, 30 mL of this solution and 170 mg of 10% palladium / carbon (50% wet product, manufactured by SIGMA-ALDRICH) were added, and the mixture was stirred at room temperature for 180 minutes in an air atmosphere. After the catalyst was filtered off, 463 mg of dithiothreitol was added to the filtrate, and the mixture was stirred at room temperature for 45 minutes. A 1 M aqueous sodium hydroxide solution was added to the reaction solution to adjust the pH to 6. The reaction mixture was extracted 5 times with 50 mL of ethyl acetate, and the ethyl acetate layer was dehydrated with sodium sulfate. After the sodium sulfate was filtered off, the filtrate was concentrated to dryness with a rotary evaporator. After dissolving 1.5 g of the ethyl acetate fraction in 5 mL of methanol, it was subjected to TOYOPEARL HW-40S column chromatography (40 mm × 450 mm, mobile phase: methanol, flow rate: 15 mL / min) to obtain 374 mg of crude teasinencin A. The obtained crude teacinencin A was dissolved in 15 mL of 5% acetonitrile (water / acetonitrile = 9.5 / 5, volume ratio), and then MCIGEL-CHP55Y column chromatography (20 mm × 300 mm, manufactured by Mitsubishi Chemical Corporation, mobile phase: water / acetonitrile). Purification by / formic acid = 870/130/1 (volume ratio), flow velocity: 7.5 mL / min) gave 286 mg of acetonitrile.
[製造例4]テアシネンシンBの合成
0.6gの(-)-EGC(三井農林株式会社製)と、0.9gの(-)-EGCgを超純水60mLとMcIlvaine緩衝液(pH5)60mLを用いて溶解した。50mLコニカルビーカー4つそれぞれにこの溶液30mLと10%パラジウム/炭素(50%水湿潤品)250mgを加えて、空気雰囲気下室温で210分間撹拌した。触媒をろ別した後、ろ液にトリス(2-カルボキシエチル)ホスフィン塩酸塩860mgを加え、室温で10分間撹拌した。反応液を直接DIAION HP-20カラムクロマトグラフィー(30mm×140mm)に供し、超純水350mLで洗浄後、40%メタノール(水/メタノール=6/4、体積比)500mLで溶出した。濃縮乾固した40%メタノール画分1.1gをメタノール5mLで溶解後、TOYOPEARL HW-40Sカラムクロマトグラフィー(40mm×450mm、移動相:メタノール、流速:15mL/min)に供し、粗テアシネンシンBを294mg得た。得られた粗テアシネンシンBを5%アセトニトリル(水/アセトニトリル=9.5/5、体積比)7.5mLに溶解後、MCIGEL-CHP55Yカラムクロマトグラフィー(20mm×300mm、移動相:水/アセトニトリル/ギ酸=900/100/1(体積比)、流速:7.5mL/min)にて精製し、テアシネンシンBを188mg得た。
[Production Example 4] Synthesis of teasinencin B
0.6 g of (-)-EGC (manufactured by Mitsui Norin Co., Ltd.) and 0.9 g of (-)-EGCg were dissolved with 60 mL of ultrapure water and 60 mL of McIlvaine buffer (pH 5). To each of the four 50 mL conical beakers, 30 mL of this solution and 250 mg of 10% palladium / carbon (50% water-wet product) were added, and the mixture was stirred at room temperature for 210 minutes in an air atmosphere. After the catalyst was filtered off, 860 mg of tris (2-carboxyethyl) phosphine hydrochloride was added to the filtrate, and the mixture was stirred at room temperature for 10 minutes. The reaction solution was directly subjected to DIAION HP-20 column chromatography (30 mm × 140 mm), washed with 350 mL of ultrapure water, and eluted with 500 mL of 40% methanol (water / methanol = 6/4, volume ratio). After dissolving 1.1 g of the concentrated and dried 40% methanol fraction in 5 mL of methanol, it was subjected to TOYOPEARL HW-40S column chromatography (40 mm × 450 mm, mobile phase: methanol, flow rate: 15 mL / min) to obtain 294 mg of crude teasinencin B. It was. After dissolving the obtained crude teacinencin B in 7.5 mL of 5% acetonitrile (water / acetonitrile = 9.5 / 5, volume ratio), MCIGEL-CHP55Y column chromatography (20 mm × 300 mm, mobile phase: water / acetonitrile / formic acid = 900 / Purification was carried out at 100/1 (volume ratio), flow velocity: 7.5 mL / min) to obtain 188 mg of acetonitrile.
[製造例5]テアシネンシンCの合成
1.2gの(-)-EGCを、超純水60mLとMcIlvaine緩衝液(pH5)60mLを用いて溶解した。50mLコニカルビーカー4つそれぞれにこの溶液30mLと10%パラジウム/炭素(50%水湿潤品)170mgを加えて、空気雰囲気下室温で240分間撹拌した。触媒をろ別した後、ろ液にトリス(2-カルボキシエチル)ホスフィン塩酸塩860mgを加え、室温で10分間撹拌した。反応液をMCIGEL-CHP55Yカラムクロマトグラフィー(20mm×300mm、移動相:水/メタノール/ギ酸=950/50/1(体積比)、流速:7.5mL/min)にて精製し、テアシネンシンCを369mg得た。
[Production Example 5] Synthesis of teasinencin C
1.2 g of (-)-EGC was dissolved in 60 mL of ultrapure water and 60 mL of McIlvaine buffer (pH 5). 30 mL of this solution and 170 mg of 10% palladium / carbon (50% wet product) were added to each of four 50 mL conical beakers, and the mixture was stirred at room temperature for 240 minutes in an air atmosphere. After the catalyst was filtered off, 860 mg of tris (2-carboxyethyl) phosphine hydrochloride was added to the filtrate, and the mixture was stirred at room temperature for 10 minutes. The reaction mixture was purified by MCIGEL-CHP55Y column chromatography (20 mm × 300 mm, mobile phase: water / methanol / formic acid = 950/50/1 (volume ratio), flow velocity: 7.5 mL / min) to obtain 369 mg of teacinencin C. It was.
[試験例1]SARSコロナウイルスに対する増殖阻害活性の測定
製造例1〜5で得られた画分、プロアントシアニジン類、テアシネンシン類のSARSコロナウイルスに対する増殖阻害活性の測定を行った。また、カテキン類((-)-EC、(-)-EGC、(-)-ECg、(-)-EGCg、全て三井農林株式会社製)についても活性の測定を行った。SARSコロナウイルスはFFM-1株(Dr. H.W. Doerr,Frankfurt University of Medicine,Germanyより分与)を用いた。培養細胞はVero細胞(アフリカミドリザル腎臓由来)を用い、培地には10重量%ウシ胎児血清、ストレプトマイシン(100μg/mL)、ペニシリン(100U/mL)を添加したダルベッコ変法イーグル最小必須培地(SIGMA-ALDRICH社製)を用いて5体積%濃度のCO2存在下、37℃で培養した。
[Test Example 1] Measurement of growth inhibitory activity against SARS coronavirus The growth inhibitory activity of the fractions, proanthocyanidins, and teacinencins obtained in Production Examples 1 to 5 against SARS coronavirus was measured. The activity of catechins ((-)-EC, (-)-EGC, (-)-ECg, (-)-EGCg, all manufactured by Mitsui Norin Co., Ltd.) was also measured. For SARS coronavirus, FFM-1 strain (distributed from Dr. HW Doerr, Frankfurt University of Medicine, Germany) was used. Vero cells (derived from African green monkey kidney) were used as the cultured cells, and 10 wt% fetal bovine serum, streptomycin (100 μg / mL), and penicillin (100 U / mL) were added to the medium, and Dalveco's modified Eagle's minimum essential medium (SIGMA-). The cells were cultured at 37 ° C. in the presence of CO 2 at a concentration of 5% by volume using ALDRICH).
6穴プレート上で90%単層を形成したVero細胞に、SARSコロナウイルスのストック液0.2mLを添加して25℃で60分間感染させた。次いで、被験物質を培養液1mL当たり0、1、3、10、30、100μg/mL(ポリフェノンGの場合のみ0、10、30、100、200、400μg/mL)となるように加え、1重量%メチルセルロースを加えたダルベッコ変法イーグル最小必須培地(5%ウシ胎児血清含有)で4日間培養した。培養後、メチルセルロースを取り除き、Vero細胞を2.5重量%クリスタルバイオレットで染色し、0.05Mリン酸緩衝生理食塩水(Mg2+及びCa2+不含、0.15M塩化ナトリウム、pH7.0)で3回洗浄した。形成されたプラーク数から1mL当たりのウイルス量をPFU(Plaque Forming Unit)/mLとして求め、さらに被験物質のIC50(ウイルスの増殖を50%阻害する濃度(阻害数から算出))を算出し、表1及び表2に示した。 Vero cells forming a 90% monolayer on a 6-well plate were infected with 0.2 mL of SARS coronavirus stock solution at 25 ° C. for 60 minutes. Then, the test substance was added so as to be 0, 1, 3, 10, 30, 100 μg / mL (0, 10, 30, 100, 200, 400 μg / mL only in the case of polyphenone G) per 1 mL of the culture medium, and 1 weight was added. The cells were cultured in Dalveco's modified Eagle's minimum essential medium (containing 5% fetal bovine serum) containing% methylcellulose for 4 days. After culturing, methylcellulose was removed, Vero cells were stained with 2.5 wt% crystal violet, and 3 times with 0.05M phosphate buffered saline (Mg 2+ and Ca 2+ free, 0.15M sodium chloride, pH 7.0). Washed. From the number of plaques formed, the amount of virus per 1 mL was calculated as PFU (Plaque Forming Unit) / mL, and the IC 50 (concentration that inhibits virus growth by 50% (calculated from the number of inhibition)) of the test substance was calculated. It is shown in Table 1 and Table 2.
緑茶抽出物粉末であるポリフェノンGに含まれるSARSコロナウイルスの増殖阻害成分の特定を行った(調製方法は製造例1を参照)。DIAION HP20を用い、ポリフェノンG(IC50=150μg/mL)を6つの画分(水画分、20%・40%・60%・80%メタノール画分、アセトン画分)に分画した。次いで、SARSコロナウイルスに対する増殖阻害活性が最も高かった40%メタノール画分(IC50=25μg/mL)を、TOYOPEARL HW-40Cにて3つの画分(画分1〜3)に分画した。次いで、増殖阻害活性が最も高かった画分3(IC50=15μg/mL)を、Mightysil RP-18 GP IIを用いた分取HPLCにて8つの画分(画分3-1〜3-8)に分画した。さらに、増殖阻害活性が高い3つの画分、画分3-2(IC50=20μg/mL)、画分3-3(IC50=20μg/mL)、画分3-4(IC50=15μg/mL)を同様の分取HPLCにて精製を行った。画分3-2より(-)-EGC-(4β→8)-(-)-EGCg及び(+)-GC-(4α→8)-(-)-EGCgを、画分3-3より(+)-GC-(4α→8)-(-)-ECgを、画分3-4より(-)-EGCg-(4β→8)-(-)-EGCgを得た。 The growth-inhibiting component of SARS coronavirus contained in polyphenone G, which is a green tea extract powder, was identified (see Production Example 1 for the preparation method). Using DIAION HP20, polyphenone G (IC 50 = 150 μg / mL) was fractionated into 6 fractions (water fraction, 20%, 40%, 60%, 80% methanol fraction, acetone fraction). Next, the 40% methanol fraction (IC 50 = 25 μg / mL), which had the highest growth inhibitory activity against SARS coronavirus, was fractionated into three fractions (fractions 1 to 3) with TOYOPEARL HW-40C. Next, fraction 3 (IC 50 = 15 μg / mL) with the highest growth inhibitory activity was subjected to preparative HPLC using Mightysil RP-18 GP II, and 8 fractions (fractions 3-1 to 3-8). ). In addition, three fractions with high growth inhibitory activity, fraction 3-2 (IC 50 = 20 μg / mL), fraction 3-3 (IC 50 = 20 μg / mL), fraction 3-4 (IC 50 = 15 μg) / mL) was purified by the same preparative HPLC. From fraction 3-2 (-)-EGC- (4β → 8)-(-)-EGCg and (+)-GC-(4α → 8)-(-)-EGCg from fraction 3-3 ( +)-GC-(4α → 8)-(-)-ECg was obtained from fractions 3-4, and (-)-EGCg-(4β → 8)-(-)-EGCg was obtained.
これらの単離した化合物は、プロアントシアニジン類の二量体に没食子酸が結合したプロアントシアニジンガレート類であった。プロアントシアニジンガレート類のSARSコロナウイルスに対する増殖阻害活性を、単量体であるカテキン類や、烏龍茶や紅茶に含まれる異なる結合様式を持つ二量体であるテアシネンシン類の活性と比較した(表2)。 These isolated compounds were proanthocyanidin gallates in which gallic acid was bound to a dimer of proanthocyanidins. The growth-inhibitory activity of proanthocyanidins against SARS coronavirus was compared with the activity of monomeric catechins and dimers of oolong tea and black tea with different binding modes (Table 2). ..
表2に示すように、単量体であるカテキン類の内、(-)-ECと(-)-EGCではIC50は100μg/mLを超え、SARSコロナウイルスに対する増殖阻害活性は認められなかった。また、単量体であるカテキン類の内、ガロイル基を有する(-)-ECgと(-)-EGCgは1〜30μg/mLの試験濃度ではウイルスの増殖阻害率が50%より低く、100μg/mLでは細胞変性が認められたためIC50の算出ができなかった。 As shown in Table 2, among the monomeric catechins, IC 50 exceeded 100 μg / mL in (-)-EC and (-)-EGC, and no growth inhibitory activity against SARS coronavirus was observed. .. Among the monomeric catechins, (-)-ECg and (-)-EGCg, which have a galloyl group, have a virus growth inhibition rate lower than 50% at a test concentration of 1 to 30 μg / mL, and 100 μg / IC 50 could not be calculated because cell degeneration was observed in mL.
一方、二量体であるプロアントシアニジン類、テアシネンシン類ではSARSコロナウイルスに対する増殖阻害活性が認められ、特にガロイル基を有するプロアントシアニジンガレート類、テアシネンシンガレート類で高い増殖阻害活性が認められた。ガロイル基を持たないプロアントシアニジン類である(-)-EGC-(4β→8)-(-)-EGCに対し、プロアントシアニジンガレート類((-)-EGC-(4β→8)-(-)-EGCg、(+)-GC-(4α→8)-(-)-EGCg、(+)-GC-(4α→8)-(-)-ECg、(-)-EGCg-(4β→8)-(-)-EGCg)は2.5〜3.3倍の活性が認められた。またガロイル基を持たないテアシネンシン類であるテアシネンシンCに対し、テアシネンシンガレート類(テアシネンシンA、テアシネンシンB)は3.3〜5.0倍の活性が認められた。 On the other hand, dimers of proanthocyanidins and teacinencins were found to have a growth inhibitory activity against SARS coronavirus, and proanthocyanidins having a galloyl group and teasinensingarates were particularly highly active in inhibiting growth. Proanthocyanidins ((-)-EGC-(4β → 8)-(-)-(-)-EGC- (4β → 8)-(-)-EGC, which are proanthocyanidins without galloyl group -EGCg, (+)-GC-(4α → 8)-(-)-EGCg, (+)-GC-(4α → 8)-(-)-ECg, (-)-EGCg-(4β → 8) -(-)-EGCg) was found to be 2.5 to 3.3 times more active. In addition, the activity of teasinencin gallates (teacinencin A and teasinencin B) was 3.3 to 5.0 times higher than that of teasinencin C, which is a teasinencin having no galloyl group.
[試験例2]ネコカリシウイルスに対する増殖阻害活性の測定
プロアントシアニジンガレート類である(+)-GC-(4α→8)-(-)-EGCgとテアシネンシン類のネコカリシウイルスに対する増殖阻害活性の測定を行った。SARSコロナウイルスに代えてネコカリシウイルスF9株、Vero細胞に変えてCRFK細胞(ネコ腎臓由来)を使用した以外は試験例1と同様の方法で、ネコカリシウイルスに対する被験物質のIC50を算出した。
[Test Example 2] Measurement of growth inhibitory activity against feline calicivirus Measurement of growth inhibitory activity of proanthocyanidins gallates (+)-GC- (4α → 8)-(-)-EGCg and teacinencins against feline calicivirus Was done. The IC 50 of the test substance against feline calicivirus was calculated by the same method as in Test Example 1 except that feline calicivirus F9 strain was used instead of SARS coronavirus and CRFK cells (derived from cat kidney) were used instead of Vero cells. ..
プロアントシアニジンガレート類である(+)-GC-(4α→8)-(-)-EGCgのIC50は30μg/mL、テアシネンシンA、テアシネンシンB及びテアシネンシンCのIC50はそれぞれ20μg/mL、10μg/mL、70μg/mLであった。ガロイル基を持たないテアシネンシンCに対し、ガロイル基を持つ(+)-GC-(4α→8)-(-)-EGCg、テアシネンシンA及びテアシネンシンBは2.3〜7.0倍の強いウイルス増殖阻害活性が認められた。 A proanthocyanidin gallate compound (+) - GC- (4α → 8) - (-) - IC 50 of EGCg is 30 [mu] g / mL, Teashinenshin A, Teashinenshin B and Teashinenshin C IC 50 of each 20μg / mL, 10μg / It was mL, 70 μg / mL. Compared to teasinencin C, which does not have a galloyl group, (+)-GC- (4α → 8)-(-)-EGCg, teacinencin A and teacinencin B, which have a galloyl group, show 2.3 to 7.0 times stronger viral growth inhibitory activity. Was done.
[試験例3]インフルエンザウイルスに対する増殖阻害活性の測定
プロアントシアニジンガレート類である(+)-GC-(4α→8)-(-)-EGCgのインフルエンザウイルスに対する増殖阻害活性の測定を行った。SARSコロナウイルスに代えてA型インフルエンザウイルスPR8株、Vero細胞に代えてMDCK細胞(イヌ腎臓由来)を使用した以外は試験例1と同様の方法で、インフルエンザウイルスに対する被験物質のIC50を算出した。(+)-GC-(4α→8)-(-)-EGCgのIC50は10μg/mLであった。
[Test Example 3] Measurement of growth inhibitory activity against influenza virus The growth inhibitory activity of proanthocyanidin gallates (+)-GC- (4α → 8)-(-)-EGCg against influenza virus was measured. IC 50 of the test substance against influenza virus was calculated by the same method as in Test Example 1 except that influenza A virus PR8 strain was used instead of SARS coronavirus and MDCK cells (derived from canine kidney) were used instead of Vero cells. .. The IC 50 of (+)-GC-(4α → 8)-(-)-EGCg was 10 μg / mL.
[配合例]チュアブル錠剤
(+)-GC-(4α→8)-(-)-EGCg0.5重量部、キシリトール33.8重量部、マンニトール30.6重量部、微結晶性セルロース6.1重量部、着香料14.1重量部、ステアリン酸4.3重量部、タルク0.6重量部、ソルビトール10.0重量部を混合した粉体を錠剤プレスによって圧縮し、ウイルス増殖阻害剤を含有する錠剤を得た。
[Formulation example] Chewable tablets
(+)-GC-(4α → 8)-(-)-EGCg 0.5 parts by weight, xylitol 33.8 parts by weight, mannitol 30.6 parts by weight, microcrystalline cellulose 6.1 parts by weight, flavoring 14.1 parts by weight, stearate 4.3 parts by weight A powder obtained by mixing parts, 0.6 parts by weight of talc and 10.0 parts by weight of sorbitol was compressed by a tablet press to obtain tablets containing a virus growth inhibitor.
本発明のウイルス増殖阻害剤はウイルス感染症の予防や治療に有用である。 The virus growth inhibitor of the present invention is useful for the prevention and treatment of viral infections.
Claims (4)
(R1及びR3はそれぞれ独立に水素原子又は水酸基を表し、R1及びR3のうち少なくとも1つは水酸基である。R2及びR4は水素原子又はガロイル基を表し、R2及びR4のうち少なくとも1つはガロイル基である。)
(R5及びR6はそれぞれ独立に水素原子又はガロイル基を表す。) A virus growth inhibitor against coronavirus or calicivirus, which contains one or more selected from proanthocyanidins gallates represented by the formula (I) and teasinensins represented by the formula (II) as active ingredients.
(R 1 and R 3 independently represent a hydrogen atom or a hydroxyl group, and at least one of R 1 and R 3 is a hydroxyl group. R 2 and R 4 represent a hydrogen atom or a galloyl group, and R 2 and R At least one of 4 is a galloyl group.)
(R 5 and R 6 independently represent a hydrogen atom or a galloyl group, respectively.)
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2016157619A JP6836229B2 (en) | 2016-08-10 | 2016-08-10 | Virus growth inhibitor |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2016157619A JP6836229B2 (en) | 2016-08-10 | 2016-08-10 | Virus growth inhibitor |
Publications (2)
Publication Number | Publication Date |
---|---|
JP2018024610A JP2018024610A (en) | 2018-02-15 |
JP6836229B2 true JP6836229B2 (en) | 2021-02-24 |
Family
ID=61193603
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP2016157619A Active JP6836229B2 (en) | 2016-08-10 | 2016-08-10 | Virus growth inhibitor |
Country Status (1)
Country | Link |
---|---|
JP (1) | JP6836229B2 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP4052575A1 (en) * | 2021-03-03 | 2022-09-07 | Berkem Developpement | Use of a grape extract as a virucide against viruses from the coronavirus family |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2020196704A (en) * | 2019-05-29 | 2020-12-10 | 学校法人帝京大学 | Prophylactic and/or therapeutic agent for influenza virus infection or corona virus infection |
JP7214080B2 (en) * | 2020-10-02 | 2023-01-30 | 学校法人北里研究所 | Antiviral agent for prevention or treatment of novel coronavirus infection |
JPWO2022102590A1 (en) * | 2020-11-13 | 2022-05-19 |
Family Cites Families (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP4507027B2 (en) * | 1998-12-04 | 2010-07-21 | 明治乳業株式会社 | MMP inhibitor |
JP2005314316A (en) * | 2004-04-30 | 2005-11-10 | Kikkoman Corp | Anti-sars coronavirus agent |
CN100534426C (en) * | 2004-05-28 | 2009-09-02 | 养生堂有限公司 | Novel use of chromene compound |
JP5076136B2 (en) * | 2005-10-28 | 2012-11-21 | 国立大学法人 鹿児島大学 | Anti-inflammatory agent or food and drink having anti-inflammatory effect |
CN101756956A (en) * | 2009-03-25 | 2010-06-30 | 赵亚力 | Use of catechin for preparing retrovirus integrase inhibitors |
JP2015214501A (en) * | 2014-05-08 | 2015-12-03 | 株式会社ニッピ | INHIBITOR FOR VIRUS INTRACELLULAR GROWTH, Mn-SOD ACTIVATOR, AND ANTIVIRAL DRUG |
-
2016
- 2016-08-10 JP JP2016157619A patent/JP6836229B2/en active Active
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP4052575A1 (en) * | 2021-03-03 | 2022-09-07 | Berkem Developpement | Use of a grape extract as a virucide against viruses from the coronavirus family |
FR3120313A1 (en) * | 2021-03-03 | 2022-09-09 | Berkem Developpement | Use of a grape extract as a virucide against viruses of the coronavirus family |
US12004515B2 (en) | 2021-03-03 | 2024-06-11 | Berkem Developpement | Use of a grape extract as a virucide against viruses of the coronavirus family |
Also Published As
Publication number | Publication date |
---|---|
JP2018024610A (en) | 2018-02-15 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP6836229B2 (en) | Virus growth inhibitor | |
EP2851071B1 (en) | Use of kaempferia parviflora wall. ex. baker extracts or flavone compound for preventing or treating muscle diseases, or improving muscle function | |
EP2328575B1 (en) | A novel standardized composition, method of manufacture and use in the resolution of rna virus infection | |
US20050010040A1 (en) | Compositions enriched in phenolic compounds and methods for producing the same | |
JP5555166B2 (en) | Α-Glucosidase inhibitor containing novel epigallocatechin gallate trimer and epigallocatechin gallate polymer | |
KR20160115889A (en) | Composition for prevention, improvement or treatment of osteoporosis comprising extract of Sigesbeckia spp. | |
KR20180034030A (en) | Agent for improvement of Cathechin absorptance on the intestinal epithelium | |
JP2015214501A (en) | INHIBITOR FOR VIRUS INTRACELLULAR GROWTH, Mn-SOD ACTIVATOR, AND ANTIVIRAL DRUG | |
US20100029756A1 (en) | Novel compositions containing isolated tetrameric type a proanthocyanadin and methods of use and manufacture | |
KR101989349B1 (en) | Composition for treating and preventing viral infection comprising extracts from Camellia japonica | |
JP2018118959A (en) | Antimicrobial composition having lindera umbellate as source material | |
KR101810041B1 (en) | Composition comprising Emodin 3-O-glucoside having immunostimulating activity | |
KR102578871B1 (en) | Composition for preventing, ameliorating or treating coronavirus infectious disease comprising herbal medicine extract as effective component | |
EP4151226A1 (en) | Coronavirus therapeutic agent comprising zanthoxylum piperitum leaf extract as active ingredient | |
KR102694541B1 (en) | Antiviral agent containing phlorotannin as an active ingredient | |
JP2023070619A (en) | Coronavirus main protease inhibitor | |
KR102594706B1 (en) | Composition for preventing, ameliorating or treating coronavirus infectious disease comprising herbal medicine extract as effective component | |
US20080249163A1 (en) | Process For Producing Product Containing Proanthocyanidin in High Proportion | |
JP2024014467A (en) | SARS-CoV-2 inactivator | |
WO2011036883A1 (en) | Anti-influenza virus agent | |
JP2024074761A (en) | Tie2 activator | |
KR20240059128A (en) | Composition for preventing, ameliorating or treating coronavirus infectious disease comprising herbal medicine extract as effective component | |
KR20210139180A (en) | Therapeutic agent for coronavirus comprising Zanthoxylum piperitum leaf extract as effective component | |
JP6347734B2 (en) | Tea-derived cyclooxygenase-2 inhibitor | |
KR20150010922A (en) | Anti-obesitic composition comprising kirenol or extract of Sigesbeckia orientalis L. |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
RD02 | Notification of acceptance of power of attorney |
Free format text: JAPANESE INTERMEDIATE CODE: A7422 Effective date: 20161205 |
|
RD04 | Notification of resignation of power of attorney |
Free format text: JAPANESE INTERMEDIATE CODE: A7424 Effective date: 20161206 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A821 Effective date: 20161206 |
|
A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20190329 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A821 Effective date: 20190401 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20200507 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20200703 |
|
A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20201113 |
|
A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20201127 |
|
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: 20201225 |
|
A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20210120 |
|
R150 | Certificate of patent or registration of utility model |
Ref document number: 6836229 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |