TWI754260B - 魚針草內酯抗新型冠狀病毒之用途 - Google Patents
魚針草內酯抗新型冠狀病毒之用途 Download PDFInfo
- Publication number
- TWI754260B TWI754260B TW109113087A TW109113087A TWI754260B TW I754260 B TWI754260 B TW I754260B TW 109113087 A TW109113087 A TW 109113087A TW 109113087 A TW109113087 A TW 109113087A TW I754260 B TWI754260 B TW I754260B
- Authority
- TW
- Taiwan
- Prior art keywords
- test
- ichthyolide
- ovatodiolide
- composition
- safe
- Prior art date
Links
- KTYZKXFERQUCPX-XGKXUXTPSA-N 3,7,11,15(17)-cembratetraene-16,2:19,6-diolide Chemical compound C([C@@H]1C(=C)C(=O)O[C@H]1/C=C(C)/C1)CC(/C)=C/CCC2=C[C@@H]1OC2=O KTYZKXFERQUCPX-XGKXUXTPSA-N 0.000 title claims abstract description 18
- KTYZKXFERQUCPX-KURKYZTESA-N ovatodiolide Natural products CC1=CCCC2=C[C@@H](CC(C)=C[C@@H]3OC(=O)C(=C)[C@H]3CC1)OC2=O KTYZKXFERQUCPX-KURKYZTESA-N 0.000 title claims abstract description 18
- 150000003839 salts Chemical class 0.000 claims abstract description 10
- 150000001875 compounds Chemical class 0.000 claims abstract description 8
- 241001678559 COVID-19 virus Species 0.000 claims abstract description 7
- 208000025721 COVID-19 Diseases 0.000 claims abstract 2
- 241000711573 Coronaviridae Species 0.000 claims description 36
- 150000002596 lactones Chemical class 0.000 claims description 23
- 230000002401 inhibitory effect Effects 0.000 claims description 22
- 238000011282 treatment Methods 0.000 claims description 14
- 230000037396 body weight Effects 0.000 claims description 13
- 239000000203 mixture Substances 0.000 claims description 13
- 206010035664 Pneumonia Diseases 0.000 claims description 12
- 239000000126 substance Substances 0.000 claims description 11
- 208000015181 infectious disease Diseases 0.000 claims description 9
- 230000002458 infectious effect Effects 0.000 claims description 8
- 201000010099 disease Diseases 0.000 claims description 6
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 6
- 239000003814 drug Substances 0.000 claims description 6
- 244000166124 Eucalyptus globulus Species 0.000 claims description 3
- 229930014626 natural product Natural products 0.000 claims description 3
- 239000003960 organic solvent Substances 0.000 claims description 2
- 230000002265 prevention Effects 0.000 claims description 2
- 238000003786 synthesis reaction Methods 0.000 claims description 2
- 241000251468 Actinopterygii Species 0.000 claims 1
- 239000008194 pharmaceutical composition Substances 0.000 abstract description 7
- 239000003937 drug carrier Substances 0.000 abstract description 3
- 230000005764 inhibitory process Effects 0.000 abstract description 3
- 238000012360 testing method Methods 0.000 description 72
- 241000700159 Rattus Species 0.000 description 29
- 238000003032 molecular docking Methods 0.000 description 25
- 244000250129 Trigonella foenum graecum Species 0.000 description 21
- 235000001484 Trigonella foenum graecum Nutrition 0.000 description 21
- 235000001019 trigonella foenum-graecum Nutrition 0.000 description 21
- 210000004027 cell Anatomy 0.000 description 20
- 235000018417 cysteine Nutrition 0.000 description 19
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 19
- 102100035765 Angiotensin-converting enzyme 2 Human genes 0.000 description 17
- 108090000975 Angiotensin-converting enzyme 2 Proteins 0.000 description 17
- 102000004225 Cathepsin B Human genes 0.000 description 17
- 108090000712 Cathepsin B Proteins 0.000 description 17
- 108090000624 Cathepsin L Proteins 0.000 description 17
- 102000004172 Cathepsin L Human genes 0.000 description 17
- 238000000034 method Methods 0.000 description 17
- 102000005962 receptors Human genes 0.000 description 16
- 108020003175 receptors Proteins 0.000 description 16
- 241000700605 Viruses Species 0.000 description 15
- 230000000694 effects Effects 0.000 description 15
- 208000001528 Coronaviridae Infections Diseases 0.000 description 14
- 241000699670 Mus sp. Species 0.000 description 13
- 239000000243 solution Substances 0.000 description 13
- 230000002209 hydrophobic effect Effects 0.000 description 12
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 12
- 101710114810 Glycoprotein Proteins 0.000 description 11
- 101710167605 Spike glycoprotein Proteins 0.000 description 11
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 10
- 238000004458 analytical method Methods 0.000 description 10
- 239000000284 extract Substances 0.000 description 10
- 239000002904 solvent Substances 0.000 description 10
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 9
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 9
- -1 Iridium lactone Chemical class 0.000 description 9
- 230000008821 health effect Effects 0.000 description 9
- 210000004185 liver Anatomy 0.000 description 9
- 210000000056 organ Anatomy 0.000 description 9
- 210000001995 reticulocyte Anatomy 0.000 description 9
- 102000004190 Enzymes Human genes 0.000 description 8
- 108090000790 Enzymes Proteins 0.000 description 8
- 230000003197 catalytic effect Effects 0.000 description 8
- 229940088598 enzyme Drugs 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 8
- 229910052740 iodine Inorganic materials 0.000 description 8
- 239000011630 iodine Substances 0.000 description 8
- 210000005259 peripheral blood Anatomy 0.000 description 8
- 239000011886 peripheral blood Substances 0.000 description 8
- 208000031404 Chromosome Aberrations Diseases 0.000 description 7
- 231100000460 acute oral toxicity Toxicity 0.000 description 7
- 239000013078 crystal Substances 0.000 description 7
- 230000002441 reversible effect Effects 0.000 description 7
- 231100000039 Ames test Toxicity 0.000 description 6
- 238000010953 Ames test Methods 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 238000011047 acute toxicity test Methods 0.000 description 6
- 239000003085 diluting agent Substances 0.000 description 6
- 230000036541 health Effects 0.000 description 6
- 230000007246 mechanism Effects 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 230000035772 mutation Effects 0.000 description 6
- 239000013642 negative control Substances 0.000 description 6
- 231100000820 toxicity test Toxicity 0.000 description 6
- 231100000694 OECD Guidelines for the Testing of Chemicals Toxicity 0.000 description 5
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 5
- 230000009471 action Effects 0.000 description 5
- 239000004480 active ingredient Substances 0.000 description 5
- 150000001336 alkenes Chemical class 0.000 description 5
- 238000011161 development Methods 0.000 description 5
- 210000003743 erythrocyte Anatomy 0.000 description 5
- 238000007499 fusion processing Methods 0.000 description 5
- 238000000338 in vitro Methods 0.000 description 5
- 231100000647 material safety data sheet Toxicity 0.000 description 5
- JRZJOMJEPLMPRA-UHFFFAOYSA-N olefin Natural products CCCCCCCC=C JRZJOMJEPLMPRA-UHFFFAOYSA-N 0.000 description 5
- 230000008520 organization Effects 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 238000004088 simulation Methods 0.000 description 5
- 208000024891 symptom Diseases 0.000 description 5
- 230000001988 toxicity Effects 0.000 description 5
- 231100000419 toxicity Toxicity 0.000 description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- 108010061994 Coronavirus Spike Glycoprotein Proteins 0.000 description 4
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 4
- 241001112090 Pseudovirus Species 0.000 description 4
- 241000607142 Salmonella Species 0.000 description 4
- 230000003213 activating effect Effects 0.000 description 4
- 125000002723 alicyclic group Chemical group 0.000 description 4
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 description 4
- 238000004113 cell culture Methods 0.000 description 4
- 210000000349 chromosome Anatomy 0.000 description 4
- DDRJAANPRJIHGJ-UHFFFAOYSA-N creatinine Chemical compound CN1CC(=O)NC1=N DDRJAANPRJIHGJ-UHFFFAOYSA-N 0.000 description 4
- 230000007613 environmental effect Effects 0.000 description 4
- 239000012091 fetal bovine serum Substances 0.000 description 4
- 230000001738 genotoxic effect Effects 0.000 description 4
- 235000013402 health food Nutrition 0.000 description 4
- 210000005260 human cell Anatomy 0.000 description 4
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 4
- 210000004962 mammalian cell Anatomy 0.000 description 4
- 102000006240 membrane receptors Human genes 0.000 description 4
- 108020004084 membrane receptors Proteins 0.000 description 4
- 238000005457 optimization Methods 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 231100000331 toxic Toxicity 0.000 description 4
- 230000002588 toxic effect Effects 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- WVDDGKGOMKODPV-UHFFFAOYSA-N Benzyl alcohol Chemical compound OCC1=CC=CC=C1 WVDDGKGOMKODPV-UHFFFAOYSA-N 0.000 description 3
- 244000025254 Cannabis sativa Species 0.000 description 3
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 3
- 206010064571 Gene mutation Diseases 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 206010058667 Oral toxicity Diseases 0.000 description 3
- 244000302661 Phyllostachys pubescens Species 0.000 description 3
- 235000003570 Phyllostachys pubescens Nutrition 0.000 description 3
- 208000005374 Poisoning Diseases 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 239000006285 cell suspension Substances 0.000 description 3
- 231100000005 chromosome aberration Toxicity 0.000 description 3
- 231100000263 cytotoxicity test Toxicity 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 239000002270 dispersing agent Substances 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 231100000025 genetic toxicology Toxicity 0.000 description 3
- 230000009545 invasion Effects 0.000 description 3
- 210000003734 kidney Anatomy 0.000 description 3
- 210000000265 leukocyte Anatomy 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000007937 lozenge Substances 0.000 description 3
- 230000009456 molecular mechanism Effects 0.000 description 3
- 231100000915 pathological change Toxicity 0.000 description 3
- 230000036285 pathological change Effects 0.000 description 3
- 231100000572 poisoning Toxicity 0.000 description 3
- 230000000607 poisoning effect Effects 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 231100000279 safety data Toxicity 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 230000001225 therapeutic effect Effects 0.000 description 3
- PUPZLCDOIYMWBV-UHFFFAOYSA-N (+/-)-1,3-Butanediol Chemical compound CC(O)CCO PUPZLCDOIYMWBV-UHFFFAOYSA-N 0.000 description 2
- WHTVZRBIWZFKQO-AWEZNQCLSA-N (S)-chloroquine Chemical compound ClC1=CC=C2C(N[C@@H](C)CCCN(CC)CC)=CC=NC2=C1 WHTVZRBIWZFKQO-AWEZNQCLSA-N 0.000 description 2
- 208000030507 AIDS Diseases 0.000 description 2
- 102100036475 Alanine aminotransferase 1 Human genes 0.000 description 2
- 108010082126 Alanine transaminase Proteins 0.000 description 2
- 108010003415 Aspartate Aminotransferases Proteins 0.000 description 2
- 102000004625 Aspartate Aminotransferases Human genes 0.000 description 2
- 208000031648 Body Weight Changes Diseases 0.000 description 2
- 206010006002 Bone pain Diseases 0.000 description 2
- 229920002261 Corn starch Polymers 0.000 description 2
- CMSMOCZEIVJLDB-UHFFFAOYSA-N Cyclophosphamide Chemical compound ClCCN(CCCl)P1(=O)NCCCO1 CMSMOCZEIVJLDB-UHFFFAOYSA-N 0.000 description 2
- 208000007882 Gastritis Diseases 0.000 description 2
- 102000001554 Hemoglobins Human genes 0.000 description 2
- 108010054147 Hemoglobins Proteins 0.000 description 2
- 101000600434 Homo sapiens Putative uncharacterized protein encoded by MIR7-3HG Proteins 0.000 description 2
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 2
- 239000005089 Luciferase Substances 0.000 description 2
- 231100000426 OECD 407 Repeated Dose 28-day Oral Toxicity Study in Rodents Toxicity 0.000 description 2
- 231100000107 OECD 471 Bacterial Reverse Mutation Test Toxicity 0.000 description 2
- 231100000217 OECD 473 In vitro Mammalian Chromosome Aberration Test Toxicity 0.000 description 2
- 231100000177 OECD 474 Mammalian Erythrocyte Micronucleus Test Toxicity 0.000 description 2
- 102000035195 Peptidases Human genes 0.000 description 2
- 108091005804 Peptidases Proteins 0.000 description 2
- 102100037401 Putative uncharacterized protein encoded by MIR7-3HG Human genes 0.000 description 2
- 241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 210000004100 adrenal gland Anatomy 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 229940024606 amino acid Drugs 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 230000001093 anti-cancer Effects 0.000 description 2
- 230000000840 anti-viral effect Effects 0.000 description 2
- 239000007900 aqueous suspension Substances 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 230000004579 body weight change Effects 0.000 description 2
- 210000004556 brain Anatomy 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- WHTVZRBIWZFKQO-UHFFFAOYSA-N chloroquine Natural products ClC1=CC=C2C(NC(C)CCCN(CC)CC)=CC=NC2=C1 WHTVZRBIWZFKQO-UHFFFAOYSA-N 0.000 description 2
- 229960003677 chloroquine Drugs 0.000 description 2
- 239000008120 corn starch Substances 0.000 description 2
- 229940099112 cornstarch Drugs 0.000 description 2
- 229940109239 creatinine Drugs 0.000 description 2
- 229960004397 cyclophosphamide Drugs 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 239000006185 dispersion Substances 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 239000000839 emulsion Substances 0.000 description 2
- 235000020765 fenugreek extract Nutrition 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 210000002216 heart Anatomy 0.000 description 2
- 229940037467 helicobacter pylori Drugs 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 2
- 239000007972 injectable composition Substances 0.000 description 2
- 230000002452 interceptive effect Effects 0.000 description 2
- 238000007917 intracranial administration Methods 0.000 description 2
- 239000008101 lactose Substances 0.000 description 2
- XQPMQYSWONIJSL-UROXQCBESA-N lepidolide Chemical compound C1=C2OC(=O)C(C)=C2[C@@H](CCC(O)=O)[C@]2(C)CC[C@]3(C)[C@@H]([C@@H](\C=C\C=C(/C)C(O)=O)C)CC[C@@]3(C)[C@@H]21 XQPMQYSWONIJSL-UROXQCBESA-N 0.000 description 2
- 239000003446 ligand Substances 0.000 description 2
- 235000019359 magnesium stearate Nutrition 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 231100000062 no-observed-adverse-effect level Toxicity 0.000 description 2
- 239000003921 oil Substances 0.000 description 2
- 235000019198 oils Nutrition 0.000 description 2
- 231100000418 oral toxicity Toxicity 0.000 description 2
- PGZUMBJQJWIWGJ-ONAKXNSWSA-N oseltamivir phosphate Chemical compound OP(O)(O)=O.CCOC(=O)C1=C[C@@H](OC(CC)CC)[C@H](NC(C)=O)[C@@H](N)C1 PGZUMBJQJWIWGJ-ONAKXNSWSA-N 0.000 description 2
- 210000001672 ovary Anatomy 0.000 description 2
- 239000000546 pharmaceutical excipient Substances 0.000 description 2
- 230000000144 pharmacologic effect Effects 0.000 description 2
- 231100000614 poison Toxicity 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 230000003449 preventive effect Effects 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- RWWYLEGWBNMMLJ-MEUHYHILSA-N remdesivir Drugs C([C@@H]1[C@H]([C@@H](O)[C@@](C#N)(O1)C=1N2N=CN=C(N)C2=CC=1)O)OP(=O)(N[C@@H](C)C(=O)OCC(CC)CC)OC1=CC=CC=C1 RWWYLEGWBNMMLJ-MEUHYHILSA-N 0.000 description 2
- RWWYLEGWBNMMLJ-YSOARWBDSA-N remdesivir Chemical compound NC1=NC=NN2C1=CC=C2[C@]1([C@@H]([C@@H]([C@H](O1)CO[P@](=O)(OC1=CC=CC=C1)N[C@H](C(=O)OCC(CC)CC)C)O)O)C#N RWWYLEGWBNMMLJ-YSOARWBDSA-N 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000012827 research and development Methods 0.000 description 2
- 238000011076 safety test Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 229910002027 silica gel Inorganic materials 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 210000000952 spleen Anatomy 0.000 description 2
- 238000012453 sprague-dawley rat model Methods 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 239000000375 suspending agent Substances 0.000 description 2
- 229940061367 tamiflu Drugs 0.000 description 2
- 210000001550 testis Anatomy 0.000 description 2
- 238000004809 thin layer chromatography Methods 0.000 description 2
- 210000001541 thymus gland Anatomy 0.000 description 2
- 230000002110 toxicologic effect Effects 0.000 description 2
- 231100000027 toxicology Toxicity 0.000 description 2
- 229960005486 vaccine Drugs 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 230000003442 weekly effect Effects 0.000 description 2
- WRIDQFICGBMAFQ-UHFFFAOYSA-N (E)-8-Octadecenoic acid Natural products CCCCCCCCCC=CCCCCCCC(O)=O WRIDQFICGBMAFQ-UHFFFAOYSA-N 0.000 description 1
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 description 1
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 description 1
- 229940124321 AIDS medicine Drugs 0.000 description 1
- 208000004998 Abdominal Pain Diseases 0.000 description 1
- 206010000087 Abdominal pain upper Diseases 0.000 description 1
- 240000000782 Anisomeles indica Species 0.000 description 1
- 235000015701 Artemisia arbuscula Nutrition 0.000 description 1
- 235000002657 Artemisia tridentata Nutrition 0.000 description 1
- 235000003261 Artemisia vulgaris Nutrition 0.000 description 1
- 240000006891 Artemisia vulgaris Species 0.000 description 1
- 108010077805 Bacterial Proteins Proteins 0.000 description 1
- 229920002134 Carboxymethyl cellulose Polymers 0.000 description 1
- 102000005600 Cathepsins Human genes 0.000 description 1
- 108010084457 Cathepsins Proteins 0.000 description 1
- 206010008631 Cholera Diseases 0.000 description 1
- 206010008805 Chromosomal abnormalities Diseases 0.000 description 1
- 229920000858 Cyclodextrin Polymers 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- IGXWBGJHJZYPQS-SSDOTTSWSA-N D-Luciferin Chemical compound OC(=O)[C@H]1CSC(C=2SC3=CC=C(O)C=C3N=2)=N1 IGXWBGJHJZYPQS-SSDOTTSWSA-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
- 230000005778 DNA damage Effects 0.000 description 1
- 231100000277 DNA damage Toxicity 0.000 description 1
- CYCGRDQQIOGCKX-UHFFFAOYSA-N Dehydro-luciferin Natural products OC(=O)C1=CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 CYCGRDQQIOGCKX-UHFFFAOYSA-N 0.000 description 1
- 201000004624 Dermatitis Diseases 0.000 description 1
- 101600122786 Drosophila melanogaster Cathepsin L (isoform A) Proteins 0.000 description 1
- 241001091575 Echeveria Species 0.000 description 1
- 241001144268 Echidna Species 0.000 description 1
- BJGNCJDXODQBOB-UHFFFAOYSA-N Fivefly Luciferin Natural products OC(=O)C1CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 BJGNCJDXODQBOB-UHFFFAOYSA-N 0.000 description 1
- 238000005033 Fourier transform infrared spectroscopy Methods 0.000 description 1
- 208000005577 Gastroenteritis Diseases 0.000 description 1
- 241000590002 Helicobacter pylori Species 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 241000725303 Human immunodeficiency virus Species 0.000 description 1
- 241000713772 Human immunodeficiency virus 1 Species 0.000 description 1
- 241000207923 Lamiaceae Species 0.000 description 1
- 108060001084 Luciferase Proteins 0.000 description 1
- DDWFXDSYGUXRAY-UHFFFAOYSA-N Luciferin Natural products CCc1c(C)c(CC2NC(=O)C(=C2C=C)C)[nH]c1Cc3[nH]c4C(=C5/NC(CC(=O)O)C(C)C5CC(=O)O)CC(=O)c4c3C DDWFXDSYGUXRAY-UHFFFAOYSA-N 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 108010090054 Membrane Glycoproteins Proteins 0.000 description 1
- 102000012750 Membrane Glycoproteins Human genes 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 240000003433 Miscanthus floridulus Species 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 206010028400 Mutagenic effect Diseases 0.000 description 1
- 208000000112 Myalgia Diseases 0.000 description 1
- 201000009053 Neurodermatitis Diseases 0.000 description 1
- 231100000474 OECD 420 Acute Oral Toxicity - Fixed Dose Procedure Toxicity 0.000 description 1
- 231100000322 OECD 423 Acute Oral toxicity - Acute Toxic Class Method Toxicity 0.000 description 1
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 description 1
- 239000005642 Oleic acid Substances 0.000 description 1
- 244000248557 Ophiopogon japonicus Species 0.000 description 1
- 241000282322 Panthera Species 0.000 description 1
- 108090000882 Peptidyl-Dipeptidase A Proteins 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 244000184734 Pyrus japonica Species 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 102000004495 STAT3 Transcription Factor Human genes 0.000 description 1
- 108010017324 STAT3 Transcription Factor Proteins 0.000 description 1
- 208000004078 Snake Bites Diseases 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 1
- 241000589970 Spirochaetales Species 0.000 description 1
- 206010070863 Toxicity to various agents Diseases 0.000 description 1
- 108050008392 Viral spike glycoproteins Proteins 0.000 description 1
- 206010047700 Vomiting Diseases 0.000 description 1
- PNNCWTXUWKENPE-UHFFFAOYSA-N [N].NC(N)=O Chemical compound [N].NC(N)=O PNNCWTXUWKENPE-UHFFFAOYSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- DPKHZNPWBDQZCN-UHFFFAOYSA-N acridine orange free base Chemical compound C1=CC(N(C)C)=CC2=NC3=CC(N(C)C)=CC=C3C=C21 DPKHZNPWBDQZCN-UHFFFAOYSA-N 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 231100000215 acute (single dose) toxicity testing Toxicity 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 231100000570 acute poisoning Toxicity 0.000 description 1
- 230000000202 analgesic effect Effects 0.000 description 1
- 239000002259 anti human immunodeficiency virus agent Substances 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000001754 anti-pyretic effect Effects 0.000 description 1
- 230000002155 anti-virotic effect Effects 0.000 description 1
- 239000002221 antipyretic Substances 0.000 description 1
- 239000003443 antiviral agent Substances 0.000 description 1
- 208000010668 atopic eczema Diseases 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- DZBUGLKDJFMEHC-UHFFFAOYSA-N benzoquinolinylidene Natural products C1=CC=CC2=CC3=CC=CC=C3N=C21 DZBUGLKDJFMEHC-UHFFFAOYSA-N 0.000 description 1
- 235000019445 benzyl alcohol Nutrition 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 239000001768 carboxy methyl cellulose Substances 0.000 description 1
- 235000010948 carboxy methyl cellulose Nutrition 0.000 description 1
- 239000008112 carboxymethyl-cellulose Substances 0.000 description 1
- 239000004359 castor oil Substances 0.000 description 1
- 235000019438 castor oil Nutrition 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 235000010980 cellulose Nutrition 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- 229940121657 clinical drug Drugs 0.000 description 1
- 238000003501 co-culture Methods 0.000 description 1
- 229940075614 colloidal silicon dioxide Drugs 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000002109 crystal growth method Methods 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 229940097362 cyclodextrins Drugs 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000002498 deadly effect Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000007791 dehumidification Methods 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 238000003255 drug test Methods 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 230000001804 emulsifying effect Effects 0.000 description 1
- 150000002170 ethers Chemical class 0.000 description 1
- 229940007062 eucalyptus extract Drugs 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 231100000024 genotoxic Toxicity 0.000 description 1
- 231100000138 genotoxicity study Toxicity 0.000 description 1
- 125000005456 glyceride group Chemical group 0.000 description 1
- ZEMPKEQAKRGZGQ-XOQCFJPHSA-N glycerol triricinoleate Natural products CCCCCC[C@@H](O)CC=CCCCCCCCC(=O)OC[C@@H](COC(=O)CCCCCCCC=CC[C@@H](O)CCCCCC)OC(=O)CCCCCCCC=CC[C@H](O)CCCCCC ZEMPKEQAKRGZGQ-XOQCFJPHSA-N 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 230000003862 health status Effects 0.000 description 1
- 238000005534 hematocrit Methods 0.000 description 1
- 241000411851 herbal medicine Species 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 206010022000 influenza Diseases 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 230000000266 injurious effect Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 238000001361 intraarterial administration Methods 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000007919 intrasynovial administration Methods 0.000 description 1
- 238000007913 intrathecal administration Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 229910052741 iridium Inorganic materials 0.000 description 1
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 210000000415 mammalian chromosome Anatomy 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 230000011278 mitosis Effects 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 208000013465 muscle pain Diseases 0.000 description 1
- 231100000243 mutagenic effect Toxicity 0.000 description 1
- 230000003505 mutagenic effect Effects 0.000 description 1
- 231100000299 mutagenicity Toxicity 0.000 description 1
- 230000007886 mutagenicity Effects 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 231100001084 no genetic toxicology Toxicity 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 239000000346 nonvolatile oil Substances 0.000 description 1
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 1
- 239000004006 olive oil Substances 0.000 description 1
- 235000008390 olive oil Nutrition 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 230000010412 perfusion Effects 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 238000005502 peroxidation Methods 0.000 description 1
- 239000000419 plant extract Substances 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 230000007096 poisonous effect Effects 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000002390 rotary evaporation Methods 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 235000019333 sodium laurylsulphate Nutrition 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 230000000087 stabilizing effect Effects 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 231100000155 toxicity by organ Toxicity 0.000 description 1
- 230000007675 toxicity by organ Effects 0.000 description 1
- 238000011269 treatment regimen Methods 0.000 description 1
- 241000712461 unidentified influenza virus Species 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 210000001835 viscera Anatomy 0.000 description 1
- 230000008673 vomiting Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 235000019786 weight gain Nutrition 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
Images
Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
本發明係關係於魚針草內酯(Ovatodiolide)安全有效使用於抑制新型冠狀病毒(SARS-CoV-2)感染的藥學組成物之用途。前述之藥學組成物，包括安全有效量之魚針草內酯或其醫藥上可接受鹽，與醫藥上可接受載體(carrier)。前述含安全有效量之魚針草內酯之藥學組成物，其頗具潛力應用於預防或治療新型冠狀病毒(SARS-CoV-2)引發的嚴重特殊傳染性肺炎(COVID-19)。
Description
本發明的重點是魚針草內酯(Ovatodiolide；Ova)抗新型冠狀病毒之用途；惟在研究開發過程中，將天然物魚針草內酯之製備鑑定分析、基本毒理試驗、魚針草內酯抗新型冠狀病毒作用機制之分子對接模擬、魚針草內酯抗新型冠狀病毒生化實驗證實等，依序進行探討並確認魚針草內酯可適量生產，安全無虞，具備抗新型冠狀病毒的作用機制與實際功效；魚針草內酯確是具有潛力的抑制新型冠狀病毒(SARS-CoV-2)感染的天然物，可用以開發為防治嚴重特殊傳染性肺炎(COVID-19)的藥物。
目前，因為全世界尚未針對新型冠狀病毒開發出有效預防疫苗或有效治療藥物；新型冠狀病毒已散佈各大洲，導致嚴重特殊傳染性肺炎正全球大流行，許多國家與各跨國藥企都在積極進行研究開發可有效防治之疫苗或藥物。
魚針草(Anisomeles indica O.Kuntze)，為台灣民間常用之草藥，又名客人抹草(廣東蕉嶺、梅縣)、金劍草、本藿香等。臺灣衛福部已將魚針草列入可供食品使用原料彙整一覽表，全株可食。魚針草為唇形科(Labiatae)之一年或越年生草本植物。魚針草主要分佈於中國西南部、印度、菲律賓、印尼爪哇及蘇門答臘，台灣全境平野至低海拔山區均可發現，
台灣花蓮玉里亦有零星藥用栽培。一般民間藥用為採集於夏、秋間，拔起全草或割取地上部位，洗淨，鮮用或曬乾用。全草有解熱、袪風、除濕、健胃、解毒、止痛、抗菌之功效。民間常用於治療感冒發熱、腹痛嘔吐、傷食霍亂、胃痛、胃腸炎、神經性皮炎、風濕骨痛、筋骨疼痛、濕疹、腫毒、瘡瘍、便毒、毒蛇咬傷。
本研發團隊長期進行魚針草育種(GenBank：GU726292)，並持續進行農場種植之魚針草全草萃取物的系列研究，尤其聚焦在魚針草內酯結晶純物質的製備(圖2)，具體執行了萃取分離純化、分析鑑定，以及抗發炎、抗病毒、抗幽門螺旋桿菌、抗癌、抗癌幹細胞等藥理作用等等研究。本團隊近年完成魚針草內酯對照治療A型與B型流行性感冒藥物「克流感」(羅氏藥廠：Tamiflu)的測試實驗，發現魚針草萃取物與魚針草內酯抑制流感病毒的良好效果。近期於2019年底獲悉，治療愛滋病藥物對治療新型冠狀病毒感染病人有呈現正面反應；按，依據文獻報導魚針草內酯可抑制HIV愛滋病毒( Fitoterapia,2000,71(5)：574-576.)。此外，既有研究亦發現魚針草內酯能抑制胃壁中幽門螺旋桿菌引起的胃炎，亦能抑制NF-B與STAT3介導的炎症反應，因此魚針草內酯亦可能緩解新型冠狀病毒引起的肺炎症狀。
本發明是基於發現魚針草內酯或其醫藥上可接受鹽，其可用於抑制新型冠狀病毒感染，甚至治療或預防嚴重特殊傳染性肺炎。特定言之，本發明係提供一種用於抑制新型冠狀病毒感染，甚至治療或預防肺炎之藥學組成物，包括安全有效量之魚針草內酯或其醫藥上可接受鹽，與醫藥上可接受載體。
本發明之魚針草內酯具有一化學結構式I，該化學結構式I如圖1所示。
本發明係提供一種以魚針草內酯為有效成分用於抑制新型冠狀病毒感染之藥學組成物為主。經模擬魚針草內酯對新型冠狀病毒表面刺突糖蛋白受體結合區(receptor-binding domain,RBD)分子對接結果顯示：魚針草內酯(Ova)結合到RBD幾個疏水氨基酸(L455,F456,Y489,F490)構成的疏水口袋，並與Y489和Q493形成氫鍵作用(圖3)。該結合位點處於新冠病毒刺突糖蛋白RBD與人體細胞膜受體血管收縮素轉換酶-2(angiotensin-converting enzyme 2,ACE2)結合的介面上，預測魚針草內酯(Ova)可阻斷或干擾病毒刺突糖蛋白受體結合區(RBD)與受體(ACE2)的直接結合。新型冠狀病毒表面刺突糖蛋白與人體細胞膜受體血管收縮素轉換酶-2(ACE2)結合是介導病毒入侵宿主的關鍵步驟，阻斷或干擾病毒與受體的結合是潛在的預防及治療策略。
同時，瞭解到宿主內體半胱氨酸蛋白水解酶Cathepsin B和Cathepsin L對新型冠狀病毒的融合過程有關鍵的作用。經模擬分子對接結果還顯示魚針草內酯(Ova)亦可能結合至內體半胱氨酸蛋白水解酶Cathepsin B和Cathepsin L的催化口袋。魚針草內酯(Ova)以疏水脂環結合Cathepsin B由Y75,P76,A173,A200，以及E245構成的疏水S2位點，並通過環外烯烴與催化半胱氨酸C29形成共價複合物從而抑制Cathepsin B的活性(圖4A)。另一方面，魚針草內酯(Ova)以疏水脂環結合Cathepsin L由L69,M70,Y72,A135,以及M161構成的疏水S2位點，並通過環外烯烴與催化半胱氨酸C25形成共價複合物從而抑制Cathepsin L的活性(圖4B)。由於內體半胱氨酸蛋白水解酶
Cathepsin B和Cathepsin L對新型冠狀病毒的融合過程有關鍵的作用，魚針草內酯(Ova)潛在地阻斷新型冠狀病毒的侵入融合過程。
本研究特別落實經由北京清華大學愛滋病綜合研究中心主任張林琦教授實驗室開發之新型冠狀病毒假病毒抑制活性檢測體系，具體評估魚針草內酯是否阻斷新型冠狀病毒感染宿主細胞過程。實驗結果顯示魚針草內酯以具體有別於氯喹(chloroquine)或瑞德西偉(Remdesivir)抑制新型冠狀病毒的分子作用機制，魚針草內酯在微摩爾級別顯著展現對新型冠狀病毒感染的抑制效果(圖5)。
魚針草內酯可擁有一或多個對掌中心，因此具有各種立體異構物形式。本發明中提及之魚針草內酯包括所有此等異構物；此外，亦包括含魚針草內酯主結構之衍生化合物，且該等衍生化合物對於抑制新型冠狀病毒的分子作用機制與本發明揭示之結合新型冠狀病毒表面刺突糖蛋白受體結合區分子對接機制，或結合宿主內體半胱氨酸蛋白水解酶Cathepsin B和Cathepsin L分子對接機制，具類同效果者。魚針草內酯具有選擇性抑制新型冠狀病毒感染的功效；由於其分子量極小，因此，可使用較低劑量的魚針草內酯或其醫藥上可接受鹽，與醫藥上可接受載體，即可得到渴望的治療效果。本發明為一抑制新型冠狀病毒感染，甚至治療或預防嚴重特殊傳染性肺炎(COVID-19)的藥學組成物，係將一安全有效量之魚針草內酯或其醫藥上可接受鹽，與醫藥上可接受載體，用於抑制新型冠狀病毒，或投予具有嚴重特殊傳染性肺炎(COVID-19)症狀的患者，以治癒、恢復、減輕、緩和、改變、治療、改善、改進或影響疾病、疾病的症狀或傾向於疾病的體質為目的。此處使用的“有效量(an effective amount)”指有效量之魚針草內酯或其醫藥上
可接受鹽，具有抑制或治療功效的量。有效量的改變是根據給藥的途徑、輔藥使用(excipient usage)以及與其他共同使用(co-usage)的活性藥劑。
此處之“嚴重特殊傳染性肺炎(COVID-19)”意指新型冠狀病毒(SARS-CoV-2)侵入人體引起的致命肺炎。新型冠狀病毒利用冠狀病毒表面的刺突醣蛋白(Spike glycoprotein)識別細胞表面的血管收縮素轉換酶-2(ACE2)，進而侵染人體的正常細胞。一種可能的機制是當病毒侵入體內後，體內的免疫細胞激烈作用，引發體內免疫風暴，釋放大量自由基(如過氧化自由)而讓蛋白質變性，DNA損傷，細胞激素過度產生，導致大量細胞的壞死，在肺部就形成了嚴重的致命肺炎。魚針草內酯可有效抑制新型冠狀病毒感染，進而預防或治療嚴重特殊傳染性肺炎(COVID-19)。
魚針草內酯是以有機溶劑萃取魚針草全草、地上部分枝葉、或葉子，並經矽膠管柱分離純化製備而得；或另以化學合成方法製備而得。例如：由“魚針草萃取”指自較適成長程度之魚針草所萃取出的魚針草萃取物。為取得該魚針草萃取物，可使用本技術領域中眾所周知的萃取技術。例如可將經乾燥與研磨之該魚針草懸浮在一溶劑或者兩種或多種溶劑之混合液於一足夠長的時間。適合的溶劑的例子包括，但不限定為：水、甲醇、乙醇、丙酮(acetone)、醚類(ether)(例如乙醚(diethyl ether))與乙酸乙酯酯類(ethyl acetate)與己烷(hexane)。之後移除固體殘餘物(例如藉由過濾)得到該魚針草萃取物溶液，其可經氧化鋁、氧化矽、矽膠管柱純化製備得魚針草內酯。
在本發明之治療方法中，魚針草內酯或其醫藥上可接受鹽類可同時給藥或分開給藥，以口服、非口服、經由吸入噴霧(inhalation spray)或
藉由植入貯存器(implanted reservoir)的方式。此處所使用之“非口服”指皮下(subcutaneous)、皮內(intracutaneous)靜脈內(intravenous)、肌肉內(intramuscular)、關節內(intraarticular)動脈(intraarterial)、滑囊(腔)內(intrasynovial)、胸骨內(intrasternal)蜘蛛膜下腔(intrathecal)、疾病部位內(intraleaional)與頭顱內(intracranial)注射以及灌注技術。本發明所使用魚針草內酯及/或其醫藥上可接受鹽類可與至少一種固體、液體或半液體狀之賦形劑或輔助劑一同形成適當的藥劑形式。其形式包括，但不限定於，藥錠、膠囊、乳劑(emulsions)、水性懸浮液(aqueous suspensions)、分散液(dispersions)與溶液。藥錠一般所使用的載體(carrier)包括乳糖與玉米澱粉。一般也將潤滑劑(lubricating agent)，例如硬脂酸鎂(magnesium stearate)加至藥錠中。用於膠囊形式的稀釋劑(diluents)包括乳糖與經乾燥的玉米澱粉。當口服給藥為水性懸浮液或乳劑時，可懸浮或溶解有效成分(active ingredient)於與乳化或懸浮劑結合的油相(oily phase)。如果需要，可加入特定甜味、調味與著色劑。本發明所使用魚針草內酯或其醫藥上可接受鹽類亦可配製成無菌注射成分(例如，水或油的懸浮液)，例如利用本技術領域中已知的技術使用適合的分散或增溼劑(例如Tween 80)與懸浮劑。無菌注射調劑也可以將無菌注射溶液或懸浮液加入無毒性非口服之稀釋劑或溶劑，例如1,3丁二醇(1,3-Butanediol)中。可使用的載具(vehicles)與溶劑包括甘露醣醇(mannitol)、水、林格氏液(Ringer’s solution)與等滲透壓氯化鈉溶液。此外，無菌、固定油常作為溶劑或懸浮媒介(例如合成的單-或雙-甘油酯(glycerides))。脂肪酸，例如油酸(oleic acid)與其甘油酯衍生物亦可用在注射劑的調製，其為天然藥學上可接受的油，例如橄欄油、蓖麻油(castor oil)，特別是於其聚氧乙基化
的(polyoxyethylated)變化形式。這些油溶液或懸浮液也可包含一長鏈醇類稀釋劑或分散劑，或者羧基甲基纖維素(carboxymethyl cellulose)或類似的分散劑。本發明所使用魚針草內酯或其醫藥上可接受鹽類亦可根據此技術領域中所熟知的技術來配製成吸入成分。例如可製成鹽類溶液，利用苯甲醇(benzyl alcohol)或其他適合的防腐劑、增強生物可利用性(bioavailability)的吸附促進劑、碳氟化合物(fluorocarbon)或其他本技術領域中熟知的助溶或分散劑來配製。用於藥學組成物的載體必須是“可接受的”，其與配方的有效成分相容(以及較佳為具有穩定有效成分之能力)以及不對病患有害。例如，助溶劑(例如環狀糊精(cyclodextrins))(其與一個或多個萃取物的活性化合物形成特定更可溶解的複合物)，為了有效成分的傳送而作為藥理學上的輔藥。其他載體的例子包括膠狀二氧化矽(colloidal silicon dioxide)、硬脂酸鎂、纖維素與烷基硫酸鹽(sodium lauryl sulfate)。
另外，由於抗病毒劑如以高劑量投予病患易產生副作用。是以依據本發明揭示之魚針草內酯系列毒理實驗結果，含對大鼠之單一劑量口服急毒性試驗、對大鼠28天餵食毒性試驗、對沙門氏菌回復突變之Ames試驗、對體外哺乳類細胞株染色體異常分析試驗、對小鼠週邊血液微核試驗等，均顯示魚針草內酯不具有基因毒性，並提供了安全口服劑量範圍。本發明醫藥組合物為含有安全有效量之魚針草內酯，用於抑制新型冠狀病毒感染，其中該安全有效量為一般成人(60公斤體重)：每日口服480毫克以內，並以持續服用28天以內為限。較佳為一般成人(60公斤體重)：每日口服20毫克至40毫克，並以持續服用7天至14天為適當。施予個別病人的特定劑量是依所有可能存在因素而定，例如：所使用之特定化合物的活性、年齡、體重、
一般健康狀況、性別、進食狀況、施用時間與路徑、排泄率、醫藥物質之組合、以及所欲治療之疾病的嚴重程度等。
本發明還提供一種組合物用於製備抑制冠狀病毒的藥物之用途，其中該組合物包括一魚針草內酯(Ovatodiolide)、或一魚針草內酯之結構異構物。
圖1.魚針草內酯(Ovatodiolide)結構式。
圖2.魚針草內酯結晶X-ray ORTEP diagram。
圖3.新型冠狀病毒突刺糖蛋白受體結合區RBD與人體細胞膜受體ACE2複合物結構，及魚針草內酯(Ova)與RBD的對接結構，魚針草內酯(Ova)結合在RBD上，干擾其與ACE2的結合。圖中標記：ACE2：血管收縮素轉換酶2；nCov S-RBD：新型冠狀病毒突刺糖蛋白受體結合區；Ovatodiolide(Ova)：魚針草內酯。
圖4.魚針草內酯(Ova)與半胱氨酸蛋白水解酶Cathepsin B(A)和Cathepsin L(B)的對接結構，其中魚針草內酯(Ova)以環外烯烴分別與Cathepsin B的催化半胱氨酸C29，以及Cathepsin L的催化半胱氨酸C25形成共價鍵。圖中標記：Ovatodiolide(Ova)：魚針草內酯；Cathepsin B：半胱氨酸蛋白水解酶B；Cathepsin L：半胱氨酸蛋白水解酶L。
圖5.魚針草內酯抑制新型冠狀病毒感染的活性曲線，IC50=3.73μM。
為了讓本發明之上述和其他目的、特徵、和優點能更明顯易懂，下文特舉較
佳實施例，做詳細說明如下：
實施例1.魚針草內酯的製備與分析：取魚針草(一年生、臺灣花蓮玉里秋季採收)大致陰乾葉子部分(800g)，放入烘箱(40℃)進行乾燥(24小時)，可得乾燥魚針草葉子(500g)。將乾燥之魚針草葉子(500g)裝入20公升桶子(PE材質)，並加入10公升95%酒精確保所有葉子均浸泡於溶劑中，之後將桶子密封保存於陰涼處7天。7天之後，將95%酒精層與葉子經由過濾分開並透過迴轉式蒸發濃縮機進行濃縮，獲得黃綠色萃取物(10g)。以氧化鋁管柱層析法進行純化(中性氧化鋁：300g)。用10mL丙酮將黃綠色萃取物(10g)溶解並加到充填好的氧化鋁管柱。溶劑沖提比例Hexane：Ethyl acetate/100%：0%，逐漸增加極性到Hexane：Ethyl acetate/70%：30%。經由薄層色層分析試紙檢測(TLC)的篩選比對，魚針草內酯可於第三個區段流出管柱。利用迴轉式蒸發濃縮機將可揮發物質抽乾後，加入15mL的丙酮回溶，利用溶劑擴散長晶方式，經過七天可得透明魚針草內酯晶體1.56g，產率約0.3%。經由X-ray,NMR,IR,Mass,HPLC等儀器鑑定。純化所得晶體確定為魚針草內酯。透過乾燥、萃取、純化和鑑定，我們成功地從魚針草中單離出魚針草內酯(ovatodiolide)，其對葉子的產率約為3000ppm，惟葉子只占魚針草全株的7%以下，且收集不易。魚針草內酯分析鑑定：X-ray Crystals of ovatodiolide are Orthorhombic,space group P212121,with a=10.7714(3),b=12.8674(3),c=13.0829(3),V=1813.29(8)Å3,D(calculated)=1.203Mg/m3,Z=4,Formula weight=328.39,Goodness-of-fit on F2=1.056,R indices(all data)：R1=0.0347,wR2=0.0942。ORTEP diagram is depicted as Figure 2(圖2).1HNMR(400MHz,CDCl3)：1.59(s,3H),1.62(m,1H),1.64(m,1H),1.72(s,3H),2.04(m,1H),
2.12(m,1H),2.19(m,1H),2.26(dd,1H),2.39(m,1H),2.45(m,1H),2.52(m,1H),2.80(m,1H),2.86(dd,1H),4.81(m,1H),4.85(bd,1H),5.08(m,1H),5.12(bd,1H),5.57(bs,1H),6.12(bs,1H),6.98(bs,1H)。13CNMR(125MHz,CDCl3)：15.1,19.3,23.7,24.9,33.3,36.3,40.3,42.7,77.9,78.8,122.9,125.0,129.1,131.2,134.3,134.5,139.6,147.4,170.4,173.0。FTIR(KBr pellet)：3100,2900,1740,1650,1430,1395,1320,1200,1110,1045,1080,980,960,930,910,880,860,820,750,625cm 1 and HRMS(ESI)m/z calcd.for C20H24O4(M+)328.1675,found 328.1672。
實施例2.魚針草內酯對大鼠之單一劑量口服急毒性試驗：本實施例為測試魚針草內酯對大鼠之單一劑量口服急毒性安全性試驗，提供食用安全評估之參考。試驗依據臺灣衛福部健康食品安全性評估-單一劑量口服急毒性試驗、美國環保署(USEPA)(Health Effects Test Guidelines,OPPTS 870.1100,Acute oral toxicity,US EPA 712-C-98-190.In：OPPTS Harmonized Test Guidelines,Series 870.3050,EPA712-C-00-366)及經濟合作暨開發組織(OECD Guidelines for the Testing of Chemicals.Section 4：Health Effects.No.420：Acute Oral Toxicity-Fixed Dose Procedure,No.423：Acute Oral Toxicity-Acute Toxic Class Method,No.425：Acute Oral Toxicity-Up and Down Method)等試驗規範進行單一劑量口服急毒性試驗。本實驗進行魚針草內酯對大鼠(Sprague-Dawley,SD品系)之單一劑量口服急毒性試驗。魚針草內酯為微黃結晶狀，試驗純度為99.95%，試驗時以10%DMSO配製成溶液濃度0.1g/mL，每隻大鼠餵食體積量為10mL/kg body weight，當日依體重經口餵食大鼠，最終投予劑量總計為1g/kg-body weight，投予後連續觀察14天。結果顯示，以魚針草內酯口服投予大鼠後，全部鼠隻皆無中毒症狀或死亡。每週體重變化
(g)方面，處理組雄鼠及雌鼠之每週體重及增重與對照組相比均無明顯差異。試驗結束後，處理組雄鼠及雌鼠之血液值變化，包括：白血球總數(WBC count)、紅血球總數(RBC count)、血球容積比(Hct)、平均紅血球體積(MCV)、平均血紅素(MCH)、平均血紅素濃度(MCHC)、血小板(platelet)總數及白血球分類等均無明顯異常。處理組雄鼠及雌鼠之血清肝腎酵素值，包括：天門冬胺酸胺基轉移酶(AST)、丙胺酸胺基轉移酶(ALT)、尿素氮(BUN)及肌酐酸(creatinine)等項目均無影響。體內臟器絕對重量(g)及臟器重量百分比方面，於腎上腺、腦、心臟、腎臟、肝臟、脾臟、胸腺、睪丸或卵巢等臟器，處理組與對照組比較並無顯著差異。經檢查體內臟器，處理組之腎上腺、腦、心臟、腎臟、肝臟、脾臟、胸腺、睪丸或卵巢等重要臟器均無肉眼病變。經組織病理檢查結果顯示，魚針草內酯處理組之重要臟器亦均無與試驗物質相關之組織病理變化。綜合試驗結果顯示，魚針草內酯1g/kg-body weight(換算人類適用劑量約為50mg/kg-body weight)之單一劑量口服急毒性試驗對大鼠並未造成急性中毒或死亡，且對體內重要臟器均未造成組織器官與毒性反應有關之病理變化。
實施例3.魚針草內酯對大鼠28天餵食毒性試驗：本實施例為測試魚針草內酯對大鼠之重複劑量口服毒性安全性試驗，藉以建立物質安全資料表(material safety data sheet)，提供人體重複服用臨床安全性評估之參考。試驗乃依據臺灣衛福部健康食品安全性評估28天餵食毒性試驗(1999)及藥品非臨床試驗安全規範(2014)，並符合美國環保署(USEPA)(Health Effects Test Guidelines,OPPTS 870.1100,Repeated Dose 28-Day Oral Toxicity Study in Rodents.In：OPPTS Harmonized Test Guidelines,Series 870.3050,EPA712-C-
00-366)及經濟合作暨開發組織(OECD Guidelines for the Testing of Chemicals.Section 4：Health Effects.No.407：Repeated Dose 28-day Oral Toxicity Study in Rodents)等試驗規範。本實驗探討魚針草內酯對人體是否可能造成潛在副作用毒性，以供臨床安全評估，進行魚針草內酯對大鼠28天重複劑量口服毒性之臨床副作用觀察試驗。魚針草內酯為微黃色結晶狀，試驗純度視為99.95%，試驗時將樣品以5% DMSO配製。大鼠(Sprague-Dawley,SD品系)分為對照組(5% DMSO)、低劑量組(10mg/kg-body weight)、中劑量組(25mg/kg body weight)及高劑量組(50mg/kg-body weight)等4組，每組20隻大鼠，雌雄鼠各半，每隻大鼠餵食體積量為10mL/kg-body weight，當日依體重經口餵食大鼠連續28天。試驗結果顯示，以魚針草內酯口服連續投予大鼠28天後，全部鼠隻皆無因試驗物質造成中毒症狀或死亡。試驗結束後，魚針草內酯各處理組雄鼠及雌鼠之體重變化、飼料消耗量、尿液、血液值、血清酵素值及臟器重量與對照組比較等數值，雖因個體差異略有上升或下降現象，但仍在正常值範圍內，或組間並不具劑量與反應相關性，並不具臨床病理意義，與試驗物質無關。檢查各組大鼠全身臟器均無明顯肉眼病理變化，經組織病理檢查結果顯示，高劑量組對體內各臟器並未造成器官毒性反應相關之病理變化。綜合以上檢查結果顯示，魚針草內酯分別以低劑量組(10mg/kg-body weight)、中劑量組(25mg/kg-body weight)及高劑量組(50mg/kg-body weight)經口連續餵食大鼠28天後，並不造成雌雄大鼠各臟器毒性反應，對大鼠28天餵食毒性試驗之「無不良影響劑量值」(No observed adverse effect level,NOAEL)為50mg/kg-body weight，換算人類適用劑量約為8mg/kg-body weight。
實施例4.魚針草內酯對沙門氏菌回復突變之Ames試驗：本實施例為測試魚針草內酯對沙門氏菌(Salmonella typhimurium)TA98、TA100、TA102、TA1535及TA1537菌株回復突變之Ames試驗，藉以建立產品安全資料(Material Safety Data Sheet)，提供使用安全評估之參考。試驗依據臺灣衛福部「健康食品安全性評估方法」之基因毒性試驗(genotoxicity study)(1999)、美國環保署(USEPA)(Health Effects Test Guidelines,Bacterial Reverse Mutation Test,US EPA 712-C-98-247.In：OPPTS Harmonized Test Guidelines,Series 870.5100,1998)及經濟合作暨開發組織(OECD Guidelines for the Testing of Chemicals.Section 4：Health Effects.No.471：Bacterial Reverse Mutation Test,2002)等試驗規範進行致變異性試驗。本實驗進行魚針草內酯對沙門氏菌(Salmonella typhimurium)TA98、TA100、TA102、TA1535及TA1537菌株回復突變之Ames試驗。試驗先以魚針草內酯之1.25、2.5及5mg/plate等濃度與菌株共同作用18-20小時，進行細菌毒性試驗。結果顯示，魚針草內酯在5mg/plate以下對TA102菌株無顯著毒性，但對TA98、TA100、TA1535及TA1537菌株具有毒性；於是再以魚針草內酯之0.63、1.25及2.5mg/plate等濃度與TA98、TA100、TA1535及TA1537菌株共同作用18小時，進行細菌毒性試驗。結果顯示，魚針草內酯在2.5mg/plate以下對TA98、TA100、TA1535及TA1537菌株皆無顯著毒性。以魚針草內酯對TA102菌株無顯著毒性之最高濃度向下連續2倍稀釋，選取0.31、0.63、1.25、2.5及5mg/plate等5個濃度作為Ames正式試驗；而TA98、TA100、TA1535及TA1537菌株無顯著毒性之最高濃度向下連續2倍稀釋，選取0.16、0.31、0.63、1.25及2.5mg/plate等5個濃度作為Ames正式試驗，分別進行魚針草內酯直接以及經大鼠肝臟活
化酵素抽出液(S9)混合後作用於沙門氏突變菌株，藉以模擬魚針草內酯經動物體內肝臟酵素(S9)代謝後之代謝產物對各菌株之基因致變異性，經共同培養48小時後計數其菌量。結果顯示，不論魚針草內酯直接或經S9作用後，對細菌回復突變菌數均未大於陰性對照組回復突變菌數2倍以上。綜合以上結果，魚針草內酯對沙門氏菌回復突變之Ames試驗並不具有致變異性，對細菌基因突變測試結果為陰性反應(non genetic mutation in Ames test)。
實施例5.魚針草內酯對體外哺乳類細胞株染色體異常分析：本實施例為測試魚針草內酯對體外哺乳類細胞株染色體異常分析，藉以建立產品安全資料(Material Safety Data Sheet)，提供使用安全評估之參考。試驗依據臺灣衛福部健康食品之體外哺乳類細胞株染色體異常分析試驗，並符合美國環保署(USEPA)(Health Effects Test Guidelines,OPPTS 870.1100,In vitro mammalian chromosome aberration test,US EPA 712-C-98-190.In：OPPTS Harmonized Test Guidelines,Series 870.3050,EPA712-C-00-366,1998)及經濟合作暨開發組織(OECD Guidelines for the Testing of Chemicals.Section 4：Health Effects.No.473：In vitro mammalian chromosome aberration test 1997)等試驗規範進行基因毒性試驗。本實驗進行魚針草內酯之細胞染色體變異試驗(Chromosomal aberration test with mammalian cell in culture)。魚針草內酯為微黃色結晶狀，試驗時以Dimethyl sulfoxide(DMSO)配製。細胞毒性試驗分為兩部分，一部分以CHO-K1細胞直接與魚針草內酯作用，另一部分以大鼠肝臟活化酵素抽出液(S9)模擬人體代謝，CHO-K1細胞與大鼠肝臟活化酵素抽出液(S9)及魚針草內酯混合後作用。於不含大鼠肝臟活化酵素抽出液(-S9)試驗先以5種測試劑量12.5、15、17.5、20及25μM進行24小時細胞毒性
試驗，結果顯示，17.5μM之魚針草內酯對CHO-K1細胞存活率約65.6%。另於含大鼠肝臟活化酵素抽出液(+S9)試驗先以5種測試劑量60、70、75、80及90μM進行24小時細胞毒性試驗，結果顯示，75μM之魚針草內酯對CHO-K1細胞存活率約60.3%，顯示魚針草內酯對CHO-K1細胞具有細胞毒性，選此濃度作為正式試驗最高劑量。細胞染色體變異試驗取樣品魚針草內酯以12.5、15與17.5μM(-S9)，配製樣品溶液於每個細胞培養皿中，共同培養24小時；另於55、65與75μM與S9共同培養3小時後，經24小時觀察細胞染色體數量及結構是否正常。結果顯示，魚針草內酯經或未經S9混合液代謝活化系統之測試條件下，在12.5、15與17.5μM(-S9)及55、65與75μM(+S9)，等3個劑量組所造成之CHO-K1細胞染色體異常頻率，與陰性對照組並無明顯增加，且對細胞染色體變異位置亦無明顯變化。綜合以上結果顯示，無論是否含有S9混合物之魚針草內酯對體外哺乳類細胞株CHO-K1之染色體均不具致變異作用。
實施例6.魚針草內酯對小鼠週邊血液微核試驗：本實施例為測試魚針草內酯之基因毒性試驗，藉以建立產品安全資料(Material Safety Data Sheet)，提供使用安全評估之參考。試驗依據臺灣衛福部健康食品安全性評估-基因毒性試驗之小鼠週邊血液微核試驗，並符合美國環保署(USEPA)(Mammalian Erythrocyte Micronucleus Test,In：OPPTS Harmonized Test Guidelines,Series 870.5395,EPA 712-C-98-226)及經濟合作暨開發組織(OECD Guidelines for the Testing of Chemicals.Section 4：Health Effects.No.474：Mammalian Erythrocyte Micronucleus Test,1997)等試驗規範進行基因毒性試驗。本實驗進行魚針草內酯對小鼠(ICR品系)週邊血液微核試驗。
本試驗主要測試魚針草內酯對嚙齒類動物體內(in vivo)週邊血液微核發生之比例，藉以評估直接或間接引發紅血球染色體或有絲分裂之基因變異造成傷害程度。試驗以ICR小鼠為試驗對象，試驗分為陰性對照組、陽性對照組(Cyclophosphamide,60mg/kg bw ip)、魚針草內酯低劑量(0.25g/kg bw)、中劑量(0.5g/kg bw)及高劑量(1g/kg bw)等5組，每組5隻小鼠(雄)，並以胃管單次餵食魚針草內酯，在投予試驗物質48小時及72小時後，評估對小鼠週邊血液之網狀紅血球及網狀紅血球中微核發生率(‰)。結果顯示，投予魚針草內酯48小時及72小時後，各處理組並無毒性症狀及體重差異。小鼠週邊血液之網狀紅血球以0.1% Acridine orange stain染色於螢光顯微鏡下呈橘紅色，網狀紅血球內可見約1/20-1/5紅血球大小黃綠色螢光之微核。比較魚針草內酯各處理組之48小時及72小時網狀紅血球數目及網狀紅血球中含微核數目均與陰性對照組間無明顯差異。陽性對照組小鼠之網狀紅血球數目與陰性對照組比較則有顯著性下降(p<0.05)，網狀紅血球中微核數目亦有顯著性增加(p<0.05)。綜合以上結果，魚針草內酯各劑量組對小鼠週邊血液之網狀紅血球和網狀紅血球中微核數目均與陰性對照組無顯著性差異，試驗結果為陰性。因此，魚針草內酯對小鼠週邊紅血球不具染色體基因變異之毒性作用。
實施例7.魚針草內酯結合新型冠狀病毒表面刺突糖蛋白受體結合區分子對接模擬研究：新型冠狀病毒表面刺突糖蛋白與人體細胞膜受體血管收縮素轉換酶2(angiotensin-converting enzyme 2,ACE2)結合是介導病毒入侵宿主的關鍵步驟，阻斷或干擾病毒與受體的結合是潛在的預防及治療策略。本實施例為基於分子對接評估魚針草內酯(Ova)是否結合新型冠狀病毒表面刺突糖蛋白以及阻斷或干擾其與受體分子血管收縮素轉換酶
2(ACE2)的結合，以闡明魚針草內酯抗病毒的機制。具體實施方法如下：取新冠病毒表面刺突糖蛋白受體結合區(receptor-binding domain,RBD)的晶體結構(PDB code：6M0J)作為分子對接受體，利用MOE軟體給RBD結構添加氫原子並進行能量優化。配體魚針草內酯(Ova)的結構亦由MOE軟體構建，採用標準MMFF94分子力場以及0.0001kcal/mol的能量梯度為收斂標準進行能量優化。基於MOE的分子對接模組進行分子對接，能量最優的對接結構進一步進行能量優化以及對接模式分析。分子對接結果顯示魚針草內酯(Ova)結合到RBD幾個疏水氨基酸(L455,F456,Y489,F490)構成的疏水口袋，並與Y489和Q493形成氫鍵作用(圖3)。該結合位點處於新冠病毒刺突糖蛋白RBD與受體ACE2結合的介面上，預測魚針草內酯(Ova)可阻斷或干擾病毒刺突糖蛋白RBD與受體ACE2的直接結合。
實施例8.魚針草內酯結合宿主內體半胱氨酸蛋白水解酶Cathepsin B和Cathepsin L分子對接模擬研究：宿主內體半胱氨酸蛋白水解酶Cathepsin B和Cathepsin L對冠狀病毒的融合過程有關鍵的作用。本實施例為基於分子對接評估魚針草內酯是否結合並抑制Cathepsin B和Cathepsin L，以闡明魚針草內酯抗病毒的作用機制。具體實施方法如下：取人內體半胱氨酸蛋白水解酶Cathepsin B和cathepsin L的晶體結構(PDB code：3AI8 & 2XU1)分別作為分子對接受體，利用MOE軟體給RBD結構添加氫原子並進行能量優化。配體魚針草內酯(Ova)的結構亦由MOE軟體構建，採用標準MMFF94分子力場以及0.0001kcal/mol的能量梯度為收斂標準進行能量優化。基於MOE的分子對接模組進行分子對接，能量最優的對接結構進一步進行能量優化以及對接模式分析。分子對接結果還顯示魚針草內酯(Ova)亦可能結合
至內體半胱氨酸蛋白水解酶Cathepsin B和Cathepsin L的催化口袋。魚針草內酯(Ova)以疏水脂環結合Cathepsin B由Y75,P76,A173,A200，以及E245構成的疏水S2位點，並通過環外烯烴與催化半胱氨酸C29形成共價複合物從而抑制Cathepsin B的活性(圖4A)。另一方面，魚針草內酯(Ova)以疏水脂環結合Cathepsin L由L69,M70,Y72,A135，以及M161構成的疏水S2位點，並通過環外烯烴與催化半胱氨酸C25形成共價複合物從而抑制Cathepsin L的活性(圖4B)。由於內體半胱氨酸蛋白水解酶Cathepsin B和Cathepsin L對冠狀病毒的融合過程有關鍵的作用，魚針草內酯(Ova)潛在地阻斷新冠病毒的侵入融合過程。
實施例9.魚針草內酯抑制新型冠狀病毒感染的活性研究：實施例7所述魚針草內酯與新型冠狀病毒表面刺突糖蛋白受體結合區(receptor-binding domain,RBD)的分子對接模擬研究結果，以及實施例8所述魚針草內酯與內體半胱氨酸蛋白水解酶Cathepsin B和Cathepsin L等的分子對接模擬研究結果，預測魚針草內酯可抑制新型冠狀病毒的感染過程。本實施例為基於北京清華大學愛滋病綜合研究中心主任張林琦教授實驗室開發之新型冠狀病毒假病毒抑制活性檢測體系，評估魚針草內酯是否阻斷新型冠狀病毒感染宿主細胞過程。具體實施方法按新型冠狀病毒假病毒構建和新型冠狀病毒感染抑制檢測兩步驟進行：步驟1.利用膜糖蛋白缺失(Env-defective)與表達螢光素蛋白的HIV-1病毒基因組質粒pNL4-3R-E-luciferase，以及表達新型冠狀病毒全長表面刺突糖蛋白質粒pcDNA3.1/SARS-CoV-2共轉染293T細胞，在含10%胎牛血清的DMEM培養基培養60小時。取培養上清液，獲得新型冠狀病毒假病毒的病毒液(簡稱SARS-CoV-2病毒液)。步驟2.取
96孔細胞培養板，每孔加入100微升魚針草內酯稀釋液和50微升SARS-CoV-2病毒液(50微升SARS-CoV-2病毒液中的病毒濃度為1×104TCID50/mL)，使得混合體系中的魚針草內酯溶液濃度為相應的稀釋濃度，37℃靜置孵育1小時。用等體積10%胎牛血清的DMEM培養基代替魚針草內酯溶液稀釋液，作為病毒對照。用等體積含10%胎牛血清的DMEM培養基代替SARS-CoV-2病毒液，作為細胞對照。取所述細胞培養板，每孔接種100微升Huh7細胞懸液(用於製備細胞懸液的溶劑為含10%胎牛血清的DMEM培養基，細胞懸液中的Huh7細胞濃度為2×105個細胞/mL)，37℃靜置孵育64小時。吸棄上清液，每孔加入150微升裂解液(微格拉斯生物技術，貨號T003，按說明書操作)，37℃靜置孵育5分鐘。取所述細胞培養板，檢測螢光素酶活性。每個處理設置多個複孔。抑制活性(%)=[1-(試驗組的螢光強度-細胞對照的螢光強度)/(病毒對照的螢光強度-細胞對照的螢光強度)]×100%。利用Prism 5軟體計算抑制活性為50%時的魚針草內酯濃度，即魚針草內酯的IC50值(圖5)。研究結果顯示魚針草內酯以具體有別於氯喹(chloroquine)或瑞德西偉(Remdesivir)抑制新型冠狀病毒之分子作用機制，在微摩爾級別顯著展現對新型冠狀病毒感染的抑制效果。
Claims (8)
- 一種組合物用於製備抑制新型冠狀病毒(SARS-CoV-2)的藥物之用途，其中該組合物包括一安全有效量之魚針草內酯(Ovatodiolide)、或一安全有效量之魚針草內酯之結構異構物，其中該魚針草內酯具有一化學結構式I：
- 如請求項1所述之用途，其中該組合物進一步包括該魚針草內酯或該魚針草內酯之結構異構物於醫藥上可接受之鹽或載體(carrier)。
- 如請求項1所述之用途，其中該組合物可用於預防或治療新型冠狀病毒(SARS-CoV-2)引起的疾病。
- 如請求項1所述之用途，其中該組合物可用於預防或治療嚴重特殊傳染性肺炎(COVID-19)。
- 如請求項1所述之用途，其中該魚針草內酯係以有機溶劑萃取魚針草，並經層析管柱分離純化製備而得之天然化合物，或係以化學合成方式製備同魚針草內酯天然物構形之人工合成化合物。
- 如請求項1所述之用途，其中該安全有效量為60公斤體重之 一般成人每日口服480毫克以內，並以持續服用28天以內為限。
- 如請求項1所述之用途，其中該安全有效量為60公斤體重之一般成人每日口服20毫克至40毫克，並持續服用7天至14天。
- 一種組合物用於製備抑制冠狀病毒的藥物之用途，其中該組合物包括一魚針草內酯(Ovatodiolide)、或一魚針草內酯之結構異構物，其中該魚針草內酯具有一化學結構式I：
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| TW109113087A TWI754260B (zh) | 2020-04-17 | 2020-04-17 | 魚針草內酯抗新型冠狀病毒之用途 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| TW109113087A TWI754260B (zh) | 2020-04-17 | 2020-04-17 | 魚針草內酯抗新型冠狀病毒之用途 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| TW202139995A TW202139995A (zh) | 2021-11-01 |
| TWI754260B true TWI754260B (zh) | 2022-02-01 |
Family
ID=80783426
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| TW109113087A TWI754260B (zh) | 2020-04-17 | 2020-04-17 | 魚針草內酯抗新型冠狀病毒之用途 |
Country Status (1)
| Country | Link |
|---|---|
| TW (1) | TWI754260B (zh) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20240408055A1 (en) * | 2021-10-21 | 2024-12-12 | Peking University Shenzhen Graduate School | Use of anisomelic acid in preparation of pharmaceutical composition for inhibiting infection and replication of sars-cov-2 and variants |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TW201313243A (zh) * | 2011-09-19 | 2013-04-01 | Yushen Biotechnology & Medical Co Ltd | 魚針草內酯或魚針草萃取物用於製造抑制細菌引起之發炎反應之醫藥組成物的用途 |
| TW201442719A (zh) * | 2013-05-14 | 2014-11-16 | Jenisa Biotechnology Corp Ltd | 魚針草萃取物及其純化物抗流感病毒之用途 |
-
2020
- 2020-04-17 TW TW109113087A patent/TWI754260B/zh active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TW201313243A (zh) * | 2011-09-19 | 2013-04-01 | Yushen Biotechnology & Medical Co Ltd | 魚針草內酯或魚針草萃取物用於製造抑制細菌引起之發炎反應之醫藥組成物的用途 |
| TW201442719A (zh) * | 2013-05-14 | 2014-11-16 | Jenisa Biotechnology Corp Ltd | 魚針草萃取物及其純化物抗流感病毒之用途 |
Non-Patent Citations (1)
| Title |
|---|
| M. Shahidul Alam et al,"HIV-inhibitory diterpenoid from Anisomeles indica", Fitoterapia, 2000, 71:574-576. * |
Also Published As
| Publication number | Publication date |
|---|---|
| TW202139995A (zh) | 2021-11-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20090111870A1 (en) | Angelicae Sinensis Extracts Useful for Treatment of Cancers | |
| US5908857A (en) | Agent for the treatment of infections | |
| KR100523562B1 (ko) | 손바닥선인장 추출물 및 이로부터 분리된 화합물을함유하는 신경세포 보호용 조성물 | |
| US20140120181A1 (en) | Composition comprising phosphatidylcholine as an active ingredient for attenuating toxicity of anticancer agent | |
| CN113521060B (zh) | 鱼针草内酯抗新型冠状病毒的用途 | |
| TWI754260B (zh) | 魚針草內酯抗新型冠狀病毒之用途 | |
| EP2116253A1 (en) | Novel phyllanthus extract | |
| US12403125B2 (en) | Use of Ovatodiolide against SARS-CoV-2 | |
| EP0297547A2 (en) | Use of hydrolyzable tannins for treatement and prophylaxis of AIDS | |
| CN1990489B (zh) | 香豆草醚类化合物及其组合物的新用途 | |
| CN117447471B (zh) | 一种吲哚二酮哌嗪类化合物及其制备方法和在制备破骨细胞分化抑制剂中的应用 | |
| CN100497279C (zh) | 一类对映桉烷醇类倍半萜抑制乙肝病毒的医药用途 | |
| US20160361334A1 (en) | Application of baicalin in preparation of drug for treating ricin poisoning | |
| CN106924243A (zh) | 梣酮在制备抗肿瘤药物增敏剂中的应用 | |
| US7744936B2 (en) | Method for preparing extract from wild ginseng showing anticancer activity and the composition comprising the same | |
| JP2002509532A (ja) | アルグラビン（ａｒｇｌａｂｉｎ）およびアルグラビン誘導体の薬学的組成物 | |
| WO2006104292A1 (en) | Pharmaceutical composition comprising arsenic acid, meta-arsenite, and pharmaceutically acceptable salts | |
| ES2341232T3 (es) | Extracto soluble en agua de una planta del genero solanum y procedimiento de preparacion del mismo, y composicion farmaceutica que contiene el extracto soluble en agua. | |
| CN112999233A (zh) | 一类来源于赤芍的单萜苷类化合物及其制法和应用 | |
| CN102727551A (zh) | 一种中药有效部位在抗hiv潜伏治疗上的应用 | |
| KR100569086B1 (ko) | 간세포 보호활성을 갖는 고로쇠 잎 추출물 및 이로부터분리된 페놀성 화합물 | |
| JP2006519752A (ja) | 慢性骨髄性白血病を治療するために有用な医薬組成物 | |
| JP2005532992A (ja) | 肝炎治療剤及び予防剤または肝臓保護剤として有用なソムオガルピ（ＡｃａｎｔｈｏｐａｎａｘＫｏｒｅａｎｕｍ）抽出物 | |
| JP4590525B2 (ja) | 抗リーシュマニア剤 | |
| CN108836975A (zh) | 野蔷薇苷的新应用 |