TWI771561B - 猴頭菇菌絲體萃取物製備用於改善中樞神經系統髓鞘化之醫藥組合物之用途 - Google Patents
猴頭菇菌絲體萃取物製備用於改善中樞神經系統髓鞘化之醫藥組合物之用途 Download PDFInfo
- Publication number
- TWI771561B TWI771561B TW108103637A TW108103637A TWI771561B TW I771561 B TWI771561 B TW I771561B TW 108103637 A TW108103637 A TW 108103637A TW 108103637 A TW108103637 A TW 108103637A TW I771561 B TWI771561 B TW I771561B
- Authority
- TW
- Taiwan
- Prior art keywords
- extract
- hericium erinaceus
- oligodendrocyte
- olg
- central nervous
- Prior art date
Links
- 239000000284 extract Substances 0.000 title claims abstract description 56
- 240000000588 Hericium erinaceus Species 0.000 title claims abstract description 53
- 235000007328 Hericium erinaceus Nutrition 0.000 title claims abstract description 52
- 210000003169 central nervous system Anatomy 0.000 title claims abstract description 19
- 239000008194 pharmaceutical composition Substances 0.000 title claims abstract description 16
- 230000023105 myelination Effects 0.000 title claims abstract description 10
- 210000004248 oligodendroglia Anatomy 0.000 claims description 61
- 210000000535 oligodendrocyte precursor cell Anatomy 0.000 claims description 40
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 34
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 24
- 230000014509 gene expression Effects 0.000 claims description 22
- KISWVXRQTGLFGD-UHFFFAOYSA-N 2-[[2-[[6-amino-2-[[2-[[2-[[5-amino-2-[[2-[[1-[2-[[6-amino-2-[(2,5-diamino-5-oxopentanoyl)amino]hexanoyl]amino]-5-(diaminomethylideneamino)pentanoyl]pyrrolidine-2-carbonyl]amino]-3-hydroxypropanoyl]amino]-5-oxopentanoyl]amino]-5-(diaminomethylideneamino)p Chemical compound C1CCN(C(=O)C(CCCN=C(N)N)NC(=O)C(CCCCN)NC(=O)C(N)CCC(N)=O)C1C(=O)NC(CO)C(=O)NC(CCC(N)=O)C(=O)NC(CCCN=C(N)N)C(=O)NC(CO)C(=O)NC(CCCCN)C(=O)NC(C(=O)NC(CC(C)C)C(O)=O)CC1=CC=C(O)C=C1 KISWVXRQTGLFGD-UHFFFAOYSA-N 0.000 claims description 20
- LEBBDRXHHNYZIA-LDUWYPJVSA-N [(2s,3r,4s,5r,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl] n-[(z)-1,3-dihydroxyoctadec-4-en-2-yl]carbamate Chemical compound CCCCCCCCCCCCC\C=C/C(O)C(CO)NC(=O)O[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O LEBBDRXHHNYZIA-LDUWYPJVSA-N 0.000 claims description 13
- 230000004069 differentiation Effects 0.000 claims description 13
- 108090000623 proteins and genes Proteins 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 10
- 101150115192 OLIG1 gene Proteins 0.000 claims description 9
- 102000004169 proteins and genes Human genes 0.000 claims description 9
- 230000006378 damage Effects 0.000 claims description 8
- 201000010099 disease Diseases 0.000 claims description 8
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 8
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 6
- 238000000855 fermentation Methods 0.000 claims description 6
- 230000004151 fermentation Effects 0.000 claims description 6
- 102000005803 Oligodendrocyte Transcription Factor 2 Human genes 0.000 claims description 5
- 108010019644 Oligodendrocyte Transcription Factor 2 Proteins 0.000 claims description 5
- 241001465754 Metazoa Species 0.000 claims description 4
- 239000007788 liquid Substances 0.000 claims description 4
- 210000003007 myelin sheath Anatomy 0.000 claims description 4
- 210000004498 neuroglial cell Anatomy 0.000 claims description 4
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 3
- 102000055324 Myelin Proteolipid Human genes 0.000 claims description 3
- 101710195940 Oligodendrocyte transcription factor 1 Proteins 0.000 claims description 3
- 102100026073 Oligodendrocyte transcription factor 1 Human genes 0.000 claims description 3
- 229940041514 candida albicans extract Drugs 0.000 claims description 3
- 239000008103 glucose Substances 0.000 claims description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 3
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 3
- 239000000401 methanolic extract Substances 0.000 claims description 3
- 239000012138 yeast extract Substances 0.000 claims description 3
- 241000196324 Embryophyta Species 0.000 claims description 2
- 101710094913 Myelin proteolipid protein Proteins 0.000 claims description 2
- 235000019764 Soybean Meal Nutrition 0.000 claims description 2
- 238000012258 culturing Methods 0.000 claims description 2
- 239000000469 ethanolic extract Substances 0.000 claims description 2
- 238000002360 preparation method Methods 0.000 claims description 2
- 239000004455 soybean meal Substances 0.000 claims description 2
- 208000027418 Wounds and injury Diseases 0.000 claims 1
- 239000006286 aqueous extract Substances 0.000 claims 1
- 208000014674 injury Diseases 0.000 claims 1
- 239000002243 precursor Substances 0.000 claims 1
- 239000013543 active substance Substances 0.000 abstract description 12
- 201000006417 multiple sclerosis Diseases 0.000 abstract description 5
- 208000015114 central nervous system disease Diseases 0.000 abstract description 4
- 208000015122 neurodegenerative disease Diseases 0.000 abstract description 4
- 230000001684 chronic effect Effects 0.000 abstract description 3
- 239000000243 solution Substances 0.000 description 19
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 18
- 102000006386 Myelin Proteins Human genes 0.000 description 18
- 108010083674 Myelin Proteins Proteins 0.000 description 18
- 210000005012 myelin Anatomy 0.000 description 18
- 102000047918 Myelin Basic Human genes 0.000 description 15
- 101710107068 Myelin basic protein Proteins 0.000 description 15
- 108020004999 messenger RNA Proteins 0.000 description 15
- NTIBMIIYTKPBNR-UHFFFAOYSA-N methoxymethyl 2-hydroxybenzoate Chemical compound COCOC(=O)C1=CC=CC=C1O NTIBMIIYTKPBNR-UHFFFAOYSA-N 0.000 description 15
- 108020004414 DNA Proteins 0.000 description 10
- 210000004027 cell Anatomy 0.000 description 9
- 230000007812 deficiency Effects 0.000 description 8
- 239000002609 medium Substances 0.000 description 7
- 101000982010 Homo sapiens Myelin proteolipid protein Proteins 0.000 description 6
- 101001129124 Mannheimia haemolytica Outer membrane lipoprotein 1 Proteins 0.000 description 6
- 102100026784 Myelin proteolipid protein Human genes 0.000 description 6
- 101000761187 Odontomachus monticola U-poneritoxin(01)-Om1a Proteins 0.000 description 6
- 238000004128 high performance liquid chromatography Methods 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 235000018102 proteins Nutrition 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- 241000123222 Hericium Species 0.000 description 5
- 230000003833 cell viability Effects 0.000 description 5
- 239000001963 growth medium Substances 0.000 description 5
- 210000002569 neuron Anatomy 0.000 description 5
- 239000000843 powder Substances 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 4
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 4
- 238000004113 cell culture Methods 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 150000002632 lipids Chemical class 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- -1 respectively Substances 0.000 description 4
- YQAQVNZGKXQJMK-ACIBSNEHSA-N (1S,2R,5R,10R,16R,19S,20R,21R)-14,16,20-trihydroxy-2,5-dimethyl-8-propan-2-yl-18,22-dioxahexacyclo[17.2.1.02,10.05,9.013,21.016,20]docosa-8,13-dien-15-one Chemical compound CC(C)C1=C2[C@H]3CCC4=C(O)C(=O)[C@@]5(O)CO[C@H]6O[C@@H]([C@@H]4[C@@]56O)[C@]3(C)CC[C@@]2(C)CC1 YQAQVNZGKXQJMK-ACIBSNEHSA-N 0.000 description 3
- 208000016192 Demyelinating disease Diseases 0.000 description 3
- 206010012305 Demyelination Diseases 0.000 description 3
- LPPCHLAEVDUIIW-NLLUTMDRSA-N Erinacine A Chemical compound O([C@H]1CC(=CC=C2C3=C(CC[C@]3(C)CC[C@]21C)C(C)C)C=O)[C@@H]1OC[C@@H](O)[C@H](O)[C@H]1O LPPCHLAEVDUIIW-NLLUTMDRSA-N 0.000 description 3
- DMPGFSQMXITJPT-ZCKYJUNOSA-N Erinacine C Chemical compound C([C@@H]1C2=C(CC[C@]2(C)CC[C@@]1(C)[C@H]1O2)C(C)C)C=C(CO)[C@H]1O[C@H]1[C@H]2OC[C@@H](O)[C@@H]1O DMPGFSQMXITJPT-ZCKYJUNOSA-N 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- PWKSKIMOESPYIA-BYPYZUCNSA-N L-N-acetyl-Cysteine Chemical compound CC(=O)N[C@@H](CS)C(O)=O PWKSKIMOESPYIA-BYPYZUCNSA-N 0.000 description 3
- 239000000020 Nitrocellulose Substances 0.000 description 3
- 230000031736 central nervous system myelination Effects 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- LPPCHLAEVDUIIW-UHFFFAOYSA-N erinacine A Natural products CC12CCC3(C)CCC(C(C)C)=C3C1=CC=C(C=O)CC2OC1OCC(O)C(O)C1O LPPCHLAEVDUIIW-UHFFFAOYSA-N 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 229920001220 nitrocellulos Polymers 0.000 description 3
- 239000001965 potato dextrose agar Substances 0.000 description 3
- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 238000012453 sprague-dawley rat model Methods 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 239000013589 supplement Substances 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- AZKSAVLVSZKNRD-UHFFFAOYSA-M 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide Chemical compound [Br-].S1C(C)=C(C)N=C1[N+]1=NC(C=2C=CC=CC=2)=NN1C1=CC=CC=C1 AZKSAVLVSZKNRD-UHFFFAOYSA-M 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- 241000282693 Cercopithecidae Species 0.000 description 2
- 108010005939 Ciliary Neurotrophic Factor Proteins 0.000 description 2
- 102100031614 Ciliary neurotrophic factor Human genes 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 102000003974 Fibroblast growth factor 2 Human genes 0.000 description 2
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- 102100034343 Integrase Human genes 0.000 description 2
- 229930040373 Paraformaldehyde Natural products 0.000 description 2
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 2
- 238000011529 RT qPCR Methods 0.000 description 2
- 239000006180 TBST buffer Substances 0.000 description 2
- 229920004890 Triton X-100 Polymers 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 210000001130 astrocyte Anatomy 0.000 description 2
- 229960002685 biotin Drugs 0.000 description 2
- 235000020958 biotin Nutrition 0.000 description 2
- 239000011616 biotin Substances 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- 210000003710 cerebral cortex Anatomy 0.000 description 2
- 230000002490 cerebral effect Effects 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- VFLDPWHFBUODDF-FCXRPNKRSA-N curcumin Chemical compound C1=C(O)C(OC)=CC(\C=C\C(=O)CC(=O)\C=C\C=2C=C(OC)C(O)=CC=2)=C1 VFLDPWHFBUODDF-FCXRPNKRSA-N 0.000 description 2
- 210000002257 embryonic structure Anatomy 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- JYGXADMDTFJGBT-VWUMJDOOSA-N hydrocortisone Chemical compound O=C1CC[C@]2(C)[C@H]3[C@@H](O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 JYGXADMDTFJGBT-VWUMJDOOSA-N 0.000 description 2
- 238000003125 immunofluorescent labeling Methods 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 239000003446 ligand Substances 0.000 description 2
- 210000001178 neural stem cell Anatomy 0.000 description 2
- 230000004770 neurodegeneration Effects 0.000 description 2
- 231100000252 nontoxic Toxicity 0.000 description 2
- 230000003000 nontoxic effect Effects 0.000 description 2
- 229920002866 paraformaldehyde Polymers 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- DAEPDZWVDSPTHF-UHFFFAOYSA-M sodium pyruvate Chemical compound [Na+].CC(=O)C([O-])=O DAEPDZWVDSPTHF-UHFFFAOYSA-M 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 238000002137 ultrasound extraction Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- 210000004885 white matter Anatomy 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- CFWRDBDJAOHXSH-SECBINFHSA-N 2-azaniumylethyl [(2r)-2,3-diacetyloxypropyl] phosphate Chemical compound CC(=O)OC[C@@H](OC(C)=O)COP(O)(=O)OCCN CFWRDBDJAOHXSH-SECBINFHSA-N 0.000 description 1
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- 206010000060 Abdominal distension Diseases 0.000 description 1
- 206010000087 Abdominal pain upper Diseases 0.000 description 1
- 235000001674 Agaricus brunnescens Nutrition 0.000 description 1
- HJCMDXDYPOUFDY-WHFBIAKZSA-N Ala-Gln Chemical compound C[C@H](N)C(=O)N[C@H](C(O)=O)CCC(N)=O HJCMDXDYPOUFDY-WHFBIAKZSA-N 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 239000012594 Earle’s Balanced Salt Solution Substances 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 208000007882 Gastritis Diseases 0.000 description 1
- 108010072051 Glatiramer Acetate Proteins 0.000 description 1
- 244000068988 Glycine max Species 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 102000014150 Interferons Human genes 0.000 description 1
- 108010050904 Interferons Proteins 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- 229930182816 L-glutamine Natural products 0.000 description 1
- 208000034800 Leukoencephalopathies Diseases 0.000 description 1
- 241000713869 Moloney murine leukemia virus Species 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 108700021862 Myelin Proteolipid Proteins 0.000 description 1
- 208000036110 Neuroinflammatory disease Diseases 0.000 description 1
- 101100228038 Rattus norvegicus Gapdh gene Proteins 0.000 description 1
- 101001120755 Rattus norvegicus Oligodendrocyte transcription factor 1 Proteins 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- 208000007107 Stomach Ulcer Diseases 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 108010076089 accutase Proteins 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 235000021120 animal protein Nutrition 0.000 description 1
- 230000002502 anti-myelin effect Effects 0.000 description 1
- 108010054176 apotransferrin Proteins 0.000 description 1
- 210000003050 axon Anatomy 0.000 description 1
- 230000003376 axonal effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 208000024330 bloating Diseases 0.000 description 1
- 244000309464 bull Species 0.000 description 1
- 230000030833 cell death Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 210000001638 cerebellum Anatomy 0.000 description 1
- 238000003501 co-culture Methods 0.000 description 1
- 230000001149 cognitive effect Effects 0.000 description 1
- 229940038717 copaxone Drugs 0.000 description 1
- 239000003246 corticosteroid Substances 0.000 description 1
- 229960001334 corticosteroids Drugs 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 229940021171 curative drug Drugs 0.000 description 1
- 229940109262 curcumin Drugs 0.000 description 1
- 235000012754 curcumin Nutrition 0.000 description 1
- 239000004148 curcumin Substances 0.000 description 1
- 230000006837 decompression Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000006735 deficit Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- VFLDPWHFBUODDF-UHFFFAOYSA-N diferuloylmethane Natural products C1=C(O)C(OC)=CC(C=CC(=O)CC(=O)C=CC=2C=C(OC)C(O)=CC=2)=C1 VFLDPWHFBUODDF-UHFFFAOYSA-N 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- BVTBRVFYZUCAKH-UHFFFAOYSA-L disodium selenite Chemical compound [Na+].[Na+].[O-][Se]([O-])=O BVTBRVFYZUCAKH-UHFFFAOYSA-L 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- MHYCRLGKOZWVEF-UHFFFAOYSA-N ethyl acetate;hydrate Chemical compound O.CCOC(C)=O MHYCRLGKOZWVEF-UHFFFAOYSA-N 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 235000013312 flour Nutrition 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 201000005917 gastric ulcer Diseases 0.000 description 1
- 235000013402 health food Nutrition 0.000 description 1
- 229960000890 hydrocortisone Drugs 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 229940079322 interferon Drugs 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 210000000274 microglia Anatomy 0.000 description 1
- 239000007758 minimum essential medium Substances 0.000 description 1
- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 230000007830 nerve conduction Effects 0.000 description 1
- 230000003959 neuroinflammation Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 235000021118 plant-derived protein Nutrition 0.000 description 1
- 108010017843 platelet-derived growth factor A Proteins 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 235000004252 protein component Nutrition 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 230000001953 sensory effect Effects 0.000 description 1
- 239000000741 silica gel Substances 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 238000010898 silica gel chromatography Methods 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 229940054269 sodium pyruvate Drugs 0.000 description 1
- 229960001471 sodium selenite Drugs 0.000 description 1
- 235000015921 sodium selenite Nutrition 0.000 description 1
- 239000011781 sodium selenite Substances 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 150000003408 sphingolipids Chemical class 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 description 1
- 238000009423 ventilation Methods 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/06—Fungi, e.g. yeasts
- A61K36/07—Basidiomycota, e.g. Cryptococcus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation or decoction
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Chemical & Material Sciences (AREA)
- Public Health (AREA)
- Neurosurgery (AREA)
- Neurology (AREA)
- Mycology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Botany (AREA)
- General Chemical & Material Sciences (AREA)
- Microbiology (AREA)
- Medical Informatics (AREA)
- Hospice & Palliative Care (AREA)
- Biotechnology (AREA)
- Alternative & Traditional Medicine (AREA)
- Psychiatry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Epidemiology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Medicines Containing Plant Substances (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Saccharide Compounds (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
本發明揭露一種猴頭菇菌絲體萃取物製備用於改善中樞神經系統髓鞘化之醫藥組合物之用途，其中萃取物包含例如猴頭素A、猴頭素C及/或猴頭素S的活性物質。中樞神經系統的髓鞘化可由包含猴頭菇活性物質之醫藥組合物加以改善，進而可治療多發性硬化症、腦白質退化症、慢性神經退化症等中樞神經疾病。
Description
本發明關於一種猴頭菇菌絲體萃取物製備用於改善中樞神經系統髓鞘化之醫藥組合物之用途。
脊椎動物之中樞神經系統包含兩類型細胞：神經元細胞(neuron)及膠質細胞(glia)，其中膠質細胞負責提供神經元細胞養分及維持微環境恆定，其細分為微膠細胞(microglia)、星狀膠質細胞(astrocyte)、及寡突膠質細胞(oligodendrocyte，OLG)。其中，寡突膠質細胞(OLG)由寡突膠質前驅細胞(oligodendrocyte precursor cell，OPC)先分化為前髓鞘化寡突膠質細胞(pre-myelinating oligodendrocyte)，再繼續分化而成。成熟的OLG延伸其觸手並纏繞神經元細胞之軸突，以形成富含脂質且多層的髓鞘(myelin)，其蛋白質成分主要為髓鞘鹼性蛋白(myelin basic protein，MBP)及髓鞘脂質蛋白(myelin proteolipid protein，PLP)。髓鞘的絕緣特性有助於神經訊息的傳導，也保護神經元細胞。當髓鞘的結構受損或脫失時，神經傳導將受阻，導致感覺、運動、認知等的缺陷。
隨著年齡增加，個體將可能因為髓鞘脫失而產生持續性的軸突缺失以及神經細胞死亡而罹患神經退化性疾病。再者，OPC更新以及分
化能力下降將致使再髓鞘化(re-myelination)的難度增加。中樞神經系統的疾病包括多發性硬化症(multiple sclerosis)、腦白質退化症(leukodystroph)、慢性神經退化症(chronic neurodegenerative disorders)等，都與OLG的髓鞘脫失或發生去髓鞘化有關，並與神經發炎相關。
以多發性硬化症為例，目前並未有可根治的藥物，急性多發性硬化症發作時通常投予皮質類固醇來治療，而一般的控制及治療則對患者皮下注射干擾素及柯珮鬆(Copaxone®)。因此，開發治療中樞神經系統的髓鞘缺失或髓鞘損傷疾病的醫藥組合物或者改善中樞神經系統髓鞘化的醫藥組合物，進而有助於治療或改善中樞神經系統的疾病確有迫切需求。
本案申請人鑑於習知技術中的不足，經過悉心試驗與研究，並一本鍥而不捨的精神，終構思出本案，能夠克服先前技術的不足，以下為本案的簡要說明。
為了開發治療中樞神經系統的髓鞘缺失或髓鞘損傷疾病的醫藥組合物，及改善中樞神經系統髓鞘化的醫藥組合物，本發明由猴頭菇(Hericium erinaceus(Bull.)Pers)菌絲體獲得天然及安全的萃取物，該萃取物包含猴頭素A(erinacine A)、猴頭素C(erinacine C)及猴頭素S(erinacine S)等活性物質。由該萃取物製備的醫藥組合物能治療中樞神經系統的髓鞘缺失或髓鞘損傷疾病，及改善中樞神經系統的髓鞘化，以有助於治療或改善中樞神經系統的疾病，例如多發性硬化症、腦白質退化症及慢性神經退化症、腦白質損傷及腦白質發炎等。此外，本發明的猴頭菇菌絲體萃取物還具有改善中樞神經系統的發炎之功效。
本發明之目的為提供一種將猴頭菇菌絲體之萃取物製備用於改善個體之中樞神經系統之髓鞘化之醫藥組合物之用途，其中萃取物包
含由猴頭素A、猴頭素C、猴頭素S及其組合所組成的群組其中之一。
在一實施例中，萃取物係以極性溶液萃取猴頭菇菌絲體而獲得，且萃取物用以增進個體之寡突膠質細胞(OLG)的寡突膠質細胞轉錄因子1(oligodendrocyte transcription factor 1，Olig1)、寡突膠質細胞轉錄因子2(oligodendrocyte transcription factor 2，Olig2)、髓鞘鹼性蛋白(MBP)、髓鞘脂質蛋白(PLP)的表現。在一實施例中，極性溶液為甲醇溶液、乙醇溶液或水。在一實施例中，當極性溶液分別為甲醇溶液、乙醇溶液及水時，萃取物分別為甲醇萃取物、乙醇溶液物及水萃取物。在一實施例中，寡突膠質細胞(OLG)係由寡突膠質前驅細胞(OPC)分化而成，且OPC用以分泌半乳糖腦苷酯(galactocerebroside，GC)。在一實施例中，萃取物用以促進OPC分化為OLG，俾補償因該中樞神經系統損傷的OLG之數量。
在一實施例中，猴頭菇菌絲體係以液態發酵培養獲得，且萃取物之作用濃度介於100ng/mL至1μg/mL之間。在一實施例中，猴頭菇菌絲體被培養於培養基，且該培養基包含葡萄糖、酵母抽出物、動植物來源蛋白質及其水解物、硫酸鎂及黃豆粉。在一實施例中，猴頭菇菌絲體在培養後經由乾燥、回溶、萃取，而得到該萃取物。
本發明還揭露一種以猴頭菇菌絲體萃取物、活性物質或所製備的醫藥組合物治療中樞神經系統之髓鞘缺失或髓鞘損傷疾病的方法，其是將醫藥上有效量的萃取物、活性物質或醫藥組合物投予所需的個體。
因此，本發明的用於改善中樞神經系統髓鞘化或者治療中樞神經系統的髓鞘缺失或髓鞘損傷疾病的醫藥組合物包括醫藥上有效量的猴頭菇菌絲體萃取物，該萃取物包含猴頭素A、猴頭素C及猴頭素S等活性物質。本發明的醫藥組合物還包括醫藥上可接受的載劑、賦形劑、稀釋劑或輔劑。本發明的醫藥組合物可以口服給藥、靜脈注射、皮下注射、腹腔注
射、肌內注射、舌下給藥等方式給予患者。
A‧‧‧猴頭素A
C‧‧‧猴頭素C
S‧‧‧猴頭素S
第1圖為猴頭素A的高效液相層析圖譜。
第2圖為猴頭素C的高效液相層析圖譜。
第3圖為猴頭素S的高效液相層析圖譜。
第4圖(A)、(B)、(C)及(D)分別為(A)猴頭菇菌絲體萃取物(HEM)、(B)猴頭素A(HeA)、(C)猴頭素C(HeC)以及(D)猴頭素S(HeS)對OPC的細胞存活率的示意圖。
第5圖(A)、(B)、(C)及(D)分別為(A)猴頭菇菌絲體萃取物(HEM)、(B)猴頭素A(HeA)、(C)猴頭素C(HeC)以及(D)猴頭素S(HeS)對OPC分化為OLG之Olig1、Olig2、MBP及PLP1 mRNA表現量的示意圖。
第6圖(A)及(B)分別為猴頭菇菌絲體萃取物(HEM)處理OPC而分化為(A)GC陽性OLG細胞以及(B)MBP陽性OLG細胞的示意圖。
第6圖(C)及(D)分別為喉頭素A(HeA)處理OPC而分化為(C)GC陽性OLG細胞以及(D)MBP陽性OLG細胞的示意圖。
第6圖(E)及(F)分別為喉頭素C(HeC)處理OPC而分化為(E)GC陽性OLG細胞以及(F)MBP陽性OLG細胞的示意圖。
第6圖(G)及(H)分別為喉頭素S(HeS)處理OPC而分化為(G)GC陽性OLG細胞以及(H)MBP陽性OLG細胞的示意圖。
本發明的上述目的及優點在參閱以下詳細說明及附隨圖式之後對那些所屬技術領域中具有通常知識者將變得更立即地顯而易見。
本發明使用猴頭菇，其主要分布於緯度較高的溫帶地區，其子實體的多醣類具有利五臟、助消化、改善消化不良、改善身體虛弱、治
療胃潰瘍、胃炎、胃痛及胃脹的功效，因此猴頭菇可作為食、藥兩用菇類。
一、實驗方法：
1.猴頭菇菌絲體的液態發酵培養
本發明實施例的猴頭菇為來自財團法人食品工業發展研究所生物資源保存及研究中心編號BCRC 35669之品種。然而，適用於本發明的猴頭菇品種不在此限。
首先，將猴頭菇菌絲體(BCRC編號：35669)無菌接種於馬鈴薯葡萄糖洋菜(potato dextrose agar，PDA)平板培養基上，於25℃培養7天。再以無菌技術刮取PDA平板培養基上之猴頭菇菌絲體，接種於裝有培養基(含2.0重量%的葡萄糖、0.1重量%的酵母抽出物、0.1重量%的動植物來源蛋白及其水解物、0.001重量%的硫酸鎂、0.1重量%的黃豆粉，並加水至100重量%)的燒瓶內，在26℃、pH 5.0、轉速120rpm震盪培養箱內震盪培養5天。之後，將燒瓶內的猴頭菇菌絲體無菌接種至含上述培養基的發酵槽內，在24℃~30℃、槽壓0.5~1.0公斤/平方公分、pH 5.0下、10-150rpm攪拌速度或不攪拌(air lift)情況，以0.5-1.0 VVM通氣速率通入空氣，培養7~10天，獲得猴頭菇菌絲體的液態培養發酵液。離心去除上清液，留下的固形物即為猴頭菇菌絲體。將此固形物冷凍乾燥獲得猴頭菇菌絲體粉末，以利於冷凍保存。
2.猴頭菇菌絲體萃取物的製備
以極性溶液萃取猴頭菇菌絲體粉末數分鐘(包含但不限於浸泡、攪拌、震盪或超音波萃取法)，再以減壓濃縮法或冷凍乾燥法進行乾燥，獲得猴頭菇菌絲體的萃取物。極性溶液包括但不限於甲醇溶液、乙醇溶液或水。當個別使用上述溶液時，分別獲得猴頭菇菌絲體的甲醇萃取物、乙醇萃取物或水萃取物。甲醇溶液為甲醇與水混合的溶液(例如1%(v/v)以
上、未滿100%(v/v))或者「純」甲醇，乙醇溶液為乙醇與水混合的溶液(例如1%(v/v)以上、未滿100%(v/v))或者「純」乙醇。
本實驗係以95%(v/v)乙醇溶液進行萃取。將猴頭菇菌絲體粉末加入其25倍重量的95%(v/v)乙醇溶液，進行第一次超音波震盪萃取1小時，將懸浮液離心獲得第一上清液。接著，將上清液以85%(v/v)乙醇溶液進行第二次超音波震盪萃取1小時，將懸浮液離心獲得第二上清液。將第二上清液減壓濃縮，獲得膏狀的猴頭菇菌絲體萃取物(簡稱萃取物)。
3.猴頭菇菌絲體萃取物的活性成分分析
將萃取物經由水-乙酸乙酯(1：1，v/v)之液液分配萃取，獲得乙酸乙酯層，再將該乙酸乙酯層以矽膠及LH-20矽膠管柱色層分析。在高效液相層析(HPLC)中，以Cosmosil® 5C18-AR-II管柱在40℃下，以起始的60%乙腈沖提，在20分鐘內逐漸提升至65%乙腈，流速1ml/min，波長為340nm。如第1圖所示，猴頭素A(erinacine A，符號A，MW：432.557g/mol)出現於7.3分鐘。在HPLC中，以Cosmosil® 5C18-AR-II管柱在40℃下，以起始的60%乙腈沖提，在20分鐘內逐漸提升至65%乙腈，流速1ml/min，波長為210nm。如第2圖所示，猴頭素C(erinacine C，符號C，MW：434.573g/mol)出現於10.4分鐘。在HPLC中，以Cosmosil® 5C18-AR-II管柱在40℃下，以起始的60%乙腈沖提，在20分鐘內逐漸提升至65%乙腈，流速1ml/min，波長為290nm。如第3圖所示，猴頭素S(erinacine S，符號S，MW：430.541g/mol)出現於14.5分鐘。猴頭素A、C及S的化學結構式如下所示。
據此，在20公噸發酵槽培養的猴頭菇菌絲體經乾燥可得約120公斤的猴頭菇菌絲體粉末。經萃取的猴頭菇菌絲體萃取物含有猴頭素A、C、S等活性物質。含有猴頭素A、C、S的猴頭菇菌絲體、粉末、萃取物可依需要製備為各式醫藥組合物或保健食品之劑型。
4.寡突膠質前驅細胞(OPC)分化成寡突膠質細胞(OLG)之建立及分析
由懷孕13.5~14.5天的Sprague-Dawley(SD)母鼠的胚胎分離其大腦皮質層，將胚胎的大腦皮質層分散後通過40μm孔徑過濾膜，將皮質層培養於未經過聚離胺酸(poly-D-lysine，PDL)處理的細胞培養皿5~7天，以形成球體狀的神經幹細胞。以Accutase細胞消化酶將神經幹細胞由細胞培養皿上分離下來，將細胞重新懸浮於生長培養液(含2%的B-27TM添加物(Gibco)、1%的N-2TM添加物(Gibco)、10ng/ml纖維母細胞生長因子2(FGF2)、10ng/ml表皮生長因子(EGF)及10ng/ml血小板衍生生長因子-AA配體(PDGF-AA ligand)的DMEM/F12培養基)並種植於經過PDL處理的細胞培養皿。2天後將生長培養液更換為分化培養液(含4mM的L-麩醯胺酸(L-glutamine)、1mM丙酮酸鈉(sodium pyruvate)、0.1%牛血清白蛋白(BSA)、50mg/ml原運鐵蛋白(apotransferrin)、5mg/ml
胰島素、30nM亞硒酸鈉、10nM生物素、10nM氫皮質酮(hydrocortisone)、15nM T3、10ng/ml毛狀神經營養因子(ciliary neurotrophic factor，CNTF)及5mg/ml N-乙醯-L半胱胺酸(N-acetyl-L-cysteine，NAC)的DMEM培養基)，並培養2天，再依實驗需求以猴頭菇菌絲體萃取物(簡稱HEM)、猴頭素A(簡稱HeA)、猴頭素C(簡稱HeC)及猴頭素S(簡稱HeS)處理。
5.寡突膠質前驅細胞(OPC)分化成寡突膠質細胞(OLG)之細胞存活率
此試驗是評估HEM、HeA、HeC、HeS是否影響OPC的存活率。首先，將2×104個OPC細胞種植於24孔培養盤並培養2天，再以不同濃度的萃取物或猴頭素處理24及48小時。加入0.5mg/ml的3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴鹽(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide，MTT)避光作用4小時，以二甲基亞碸(DMSO)溶解藍紫色結晶formazan，再以酵素免疫分析測讀儀(TECAN Sunrise ELISA Reader)測量波長595nm的吸光值，計算細胞存活率。
6. OLG的髓鞘化能力
此試驗是以定量即時聚合酶鏈反應(quantitative real-time polymerase chain reaction，Q-PCR)測定經處理後的細胞的特定基因表現量，以分析OLG的髓鞘化能力。首先，將經過猴頭菇萃取物處理後的OLG之RNA(1ug/樣本)以莫洛尼氏鼠類白血腫瘤病毒反轉錄酶(M-MLV reverse transcriptase)將其轉錄成cDNA。將cDNA、SYBR® Master Mixes及大鼠寡突膠質細胞轉錄因子1(Olig1)、寡突膠質細胞轉錄因子2(Olig2)、髓鞘鹼性蛋白(MBP)或髓鞘脂質蛋白(PLP)引子混合(參見表1)，測定相對mRNA表現量，並以大鼠GAPDH基因為控制組基因。以StepOne軟體2.12版(Applied Biosystems)分析實驗結果。
7.西方墨點法
此試驗是以習用的十二烷基硫酸鈉聚丙烯醯胺凝膠電泳(SDS-PAGE)及西方墨點法分析細胞之蛋白質表現量。簡而言之，細胞蛋白質經過SDS-PAGE後轉漬至硝化纖維膜上，加入一次抗體於低溫作用15小時以上。以TBST緩衝液清洗硝化纖維膜，再加入二次抗體於室溫作用1小時。以TBST緩衝液清洗硝化纖維膜，再以ECL進行冷光呈色，於底片顯影。
8.免疫螢光染色
以4%三聚甲醛(paraformaldehyde)固定經處理的細胞玻片15分鐘，再以0.1% Triton X100 PBS作用15分鐘。以PBS清洗3次，再加
入含有1%馬血清的抗MBP的抗體(NB1018，Calbiochem)或抗半乳糖腦苷酯(GC)的抗體(MAB342，Millipore)，於4℃隔夜作用。以PBS清洗3次，加入接有生物素之二次抗體，於室溫作用1小時。以PBS清洗3次，加入含有avidin-Cy3的三次抗體，室溫作用45分鐘。以DAPI(1μg/ml)反應1分鐘，最後以90%甘油封片，於顯微鏡(FV1000，Japan)觀察結果。
9.動物實驗模式之建立
以震動式活組織切片機(MicroslicerTM DTK-1000 vibratory tissue slicer)將出生7天的SD大鼠仔鼠的小腦切成厚度為300μm的薄片，置於插入式細胞培養皿(0.4μm Millicell® cell culture insert，Millipore)，以小腦組織切片培養液(Cerebellar slice culture medium，含50%的含厄爾鹽(Earle’s salt)的最低必需培養基(MEM)、35%厄爾平衡鹽溶液、15%熱失活的馬血清及1%的GlutaMAXTM補充劑)培養3天。以不同濃度的猴頭菇萃取物或猴頭素處理切片11天，於共同培養的第14天時使用4%三聚甲醛固定2小時，再以1% Triton-X100 PBS作用2天。以免疫螢光法進行抗髓鞘鹼性蛋白的小鼠單株抗體(anti-myelin basic protein mouse(anti-MBP)mAb，NE1018，Calbiochem)及抗神經纖維絲H的抗體(anti-neurofilament H antibody，NF200，AB5539，Millipore)的雙螢光染色，在顯微鏡(FV1000,Japan)下觀察結果。
10.統計分析
實驗數據以「平均值±標準誤」表示，以t-test分析組間差異，p<0.05為具有統計學上顯著差異。
二、實驗結果：
1. HEM、HeA、HeC及HeS對OPC的細胞存活率
請參閱第4圖(A)、(B)、(C)及(D)，其分別為(A)猴頭菇菌絲
體萃取物(HEM)、(B)猴頭素A(HeA)、(C)猴頭素C(HeC)以及(D)猴頭素S(HeS)對OPC的細胞存活率的示意圖。第4圖(A)至(D)的對照組為未加萃取物或活性物質處理的試驗。在第4圖(A)至(D)中，以10ng/ml~100μg/ml的HEM處理OPC細胞24~48小時，並不會對OPC細胞產生毒性(第4圖(A))；而0.001ng/ml~10ng/ml的HeA、HeC及HeS處理OPC細胞24~48小時亦不會對OPC細胞產生毒性(第4圖(B)、(C)及(D))。因此，可證明在適當濃度下，猴頭菇菌絲體萃取物(HEM)、活性物質(HeA、HeC及HeS)均不影響OPC的細胞存活率，對OPC是安全、無毒性的。
2. HEM、HeA、HeC及HeS對OPC分化為OLG之影響及基因表現
請參閱第5圖(A)、(B)、(C)及(D)，其分別為(A)猴頭菇菌絲體萃取物(HEM)、(B)猴頭素A(HeA)、(C)猴頭素C(HeC)以及(D)猴頭素S(HeS)對OPC分化為OLG之Olig1、Olig2、MBP及PLP1 mRNA表現量的示意圖。第5圖(A)至(D)的對照組為未以萃取物或活性物質處理的試驗。在第5圖(A)中，100ng/ml~1μg/ml的HEM增加了OLG細胞的Olig2 mRNA表現量(小圖(A)-2)，而成熟的OLG細胞的MBP及PLP1 mRNA表現量亦增加(小圖(A)-3及(A)-4)。此外，經100ng/ml及1μg/ml的HEM處理的OPC細胞，分化後的OLG細胞數目高於控制組(結果未示出)。
在第5圖(B)中，0.001ng/ml~10μg/ml的HeA同樣增加了OLG細胞的Olig2 mRNA表現量(小圖(B)-2)，HeA亦促進OLG細胞的MBP及PLP1 mRNA表現量增加(小圖(B)-3及(B)-4)。
在第5圖(C)中，不同濃度的HeC增加OLG細胞的Olig1、Olig2、MBP及PLP1 mRNA表現量，例如0.01ng/ml~10ng/ml的HeC增加OLG細胞的Olig1 mRNA表現量(小圖(C)-1)，0.1ng/ml~10ng/ml的
HeC增加OLG細胞的Olig2 mRNA表現量(小圖(C)-2)，0.01ng/ml的HeC大幅度地增加OLG細胞的MBP mRNA表現量(小圖(C)-3)，HeC對OLG細胞的PLP1 mRNA表現量並無顯著差異(小圖(C)-4)。
在第5圖(D)中，0.01ng./ml的HeS可大幅度地增加OLG細胞的Olig1、MBP、PLP mRNA表現量(小圖(D)-1、(D)-3及(D)-4)，0.001ng/ml~0.1ng/ml的HeS可增加OLG細胞的Olig2 mRNA表現量(小圖(D)-2)。0.01ng/ml的HeS可促進OLG細胞的MBP及PLP蛋白質的表現量(西方墨點法結果未示出)。
請參閱第6圖(A)至(H)，免疫螢光染色實驗結果顯示，萃取物(HEM)及活性物質(HeA、HeC及HeS)均能促進OPC分化為帶有GC陽性的OLG(第6圖(A)、(C)、(E)及(G))以及帶有MBP陽性的OLG(第6圖(B)、(D)、(F)及(H))。定量結果亦顯示，HeS促進OPC分化為帶有GC陽性的OLG以及帶有MBP陽性的OLG(第6圖(G)及(H))的能力較HeA(第6圖(C)及(D))或HeC(第6圖(E)及(F))還要好。
綜上所述，本發明的猴頭菇菌絲體萃取物及其活性物質(HeA、HeC及HeS)對寡突膠質前驅細胞(OPC)是安全、無毒性的，可促進OPC分化為寡突膠質細胞(OLG)，並促進OLG纏繞軸突的髓鞘化過程的寡突膠質細胞轉錄因子1(Olig1)、寡突膠質細胞轉錄因子2(Olig2)、髓鞘鹼性蛋白(MBP)、髓鞘脂質蛋白(PLP)的mRNA表現量及蛋白質表現量，並使OLG為成為帶有半乳糖腦苷酯(GC)陽性及帶有MBP陽性的OLG。因此，本發明的猴頭菇菌絲體萃取物及其所含的猴頭素A、C及S可被用於製備改善個體中樞神經系統髓鞘化或者治療髓鞘缺失或髓鞘損傷之疾病之醫藥組合物的用途。
本發明實屬難能的創新發明，深具產業價值，援依法提出申
請。此外，本發明可以由所屬技術領域中具有通常知識者做任何修改，但不脫離如所附申請專利範圍所要保護的範圍。
<110> 葡萄王生技股份有限公司
<120> 猴頭菇菌絲體萃取物製備用於改善中樞神經系統髓鞘化之醫藥組合物之用途
<160> 10
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213> Olig1-Forward
<400> 1
<210> 2
<211> 20
<212> DNA
<213> Olig1-Reverse
<400> 1
<210> 3
<211> 20
<212> DNA
<213> Olig2-Forward
<400> 1
<210> 4
<211> 20
<212> DNA
<213> Olig2-Reverse
<400> 1
<210> 5
<211> 20
<212> DNA
<213> MBP-Forward
<400> 1
<210> 6
<211> 20
<212> DNA
<213> MBP-Reverse
<400> 1
<210> 7
<211> 20
<212> DNA
<213> PLP-Forward
<400> 1
<210> 8
<211> 20
<212> DNA
<213> PLP-Reverse
<400> 1
<210> 9
<211> 18
<212> DNA
<213> GAPDH-Forward
<400> 1
<210> 10
<211> 18
<212> DNA
<213> GAPDH-Reverse
<400> 1
Claims (9)
- 一種將一猴頭菇菌絲體之一萃取物製備用於治療一個體之中樞神經系統之一髓鞘缺失或一髓鞘損傷之疾病及改善中樞神經系統之髓鞘化之一醫藥組合物之用途，其中該萃取物包含由猴頭素A、猴頭素C、猴頭素S及其組合所組成的群組其中之一。
- 如申請專利範圍第1項所述的用途，其中該萃取物係以一極性溶液萃取該猴頭菇菌絲體而獲得，且該萃取物用以增進該個體之寡突膠質細胞(oligodendrocyte，OLG)的寡突膠質細胞轉錄因子1(Olig1)、寡突膠質細胞轉錄因子2(Olig2)、髓鞘鹼性蛋白(myelin basic protein，MBP)、髓鞘脂質蛋白(myelin proteolipid protein，PLP)的表現。
- 如申請專利範圍第2項所述的用途，其中該極性溶液為一甲醇溶液、一乙醇溶液或水。
- 如申請專利範圍第3項所述的用途，其中當該極性溶液為該甲醇溶液時，該萃取物為一甲醇萃取物；當該極性溶液為該乙醇溶液時，該萃取物為一乙醇萃取物；且當該極性溶液為水時，該萃取物為一水萃取物。
- 如申請專利範圍第2項所述的用途，其中該寡突膠質細胞(OLG)係由寡突膠質前驅細胞(oligodendrocyte precursor cell，OPC)分化而成，且該寡突膠質前驅細胞(OPC)用以分泌半乳糖腦苷酯(galactocerebroside，GC)。
- 如申請專利範圍第5項所述的用途，其中該萃取物用以促進該寡突膠質前驅細胞(OPC)分化為該寡突膠質細胞(OLG)，俾補償因該中樞神經系統損傷的寡突膠質細胞(OLG)之數量。
- 如申請專利範圍第1項所述的用途，其中該猴頭菇菌絲體係以液態發酵培養獲得，且該萃取物之一作用濃度介於10ng/mL至100μg/mL之間。
- 如申請專利範圍第1項所述的用途，其中該猴頭菇菌絲體被培養於一培養基，且該培養基包含葡萄糖、酵母抽出物、動植物來源蛋白質及其水解物、硫酸鎂及黃豆粉。
- 如申請專利範圍第1項所述的用途，其中該猴頭菇菌絲體在培養後經由乾燥、回溶、萃取，而得到該萃取物。
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| TW108103637A TWI771561B (zh) | 2019-01-30 | 2019-01-30 | 猴頭菇菌絲體萃取物製備用於改善中樞神經系統髓鞘化之醫藥組合物之用途 |
| CN202010076480.5A CN111494435A (zh) | 2019-01-30 | 2020-01-23 | 猴头菇菌丝体萃取物制备用于改善中枢神经系统髓鞘化的医药组合物的用途 |
| JP2020012561A JP6952812B2 (ja) | 2019-01-30 | 2020-01-29 | ヘリシン・エリナセウス菌糸体抽出物によって調製する中枢神経系の髄鞘化を改善するための医薬組成物の使用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| TW108103637A TWI771561B (zh) | 2019-01-30 | 2019-01-30 | 猴頭菇菌絲體萃取物製備用於改善中樞神經系統髓鞘化之醫藥組合物之用途 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| TW202027756A TW202027756A (zh) | 2020-08-01 |
| TWI771561B true TWI771561B (zh) | 2022-07-21 |
Family
ID=71875774
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| TW108103637A TWI771561B (zh) | 2019-01-30 | 2019-01-30 | 猴頭菇菌絲體萃取物製備用於改善中樞神經系統髓鞘化之醫藥組合物之用途 |
Country Status (3)
| Country | Link |
|---|---|
| JP (1) | JP6952812B2 (zh) |
| CN (1) | CN111494435A (zh) |
| TW (1) | TWI771561B (zh) |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20040018460A (ko) * | 2001-07-16 | 2004-03-03 | 다카라 바이오 가부시키가이샤 | 치료제 |
| US7390512B2 (en) * | 2006-09-29 | 2008-06-24 | Jose Angel Olalde Rangel | Multiple sclerosis synergistic phyto-nutraceutical composition |
| CN103467544A (zh) * | 2013-09-17 | 2013-12-25 | 南京通泽农业科技有限公司 | 一种提取猴头菌素a的方法 |
| CN105287753A (zh) * | 2015-11-22 | 2016-02-03 | 青岛麦瑞特医药技术有限公司 | 一种治疗多发性硬化症的中药组合物 |
| TWI605819B (zh) * | 2015-12-14 | 2017-11-21 | Grape King Bio Ltd | Active substance for treating dementia, preparation method thereof, pharmaceutical composition containing the same, and preparation method of the pharmaceutical composition |
| US20180021326A1 (en) * | 2016-07-23 | 2018-01-25 | Paul Edward Stamets | Compositions and methods for enhancing neuroregeneration and cognition by combining mushroom extracts containing active ingredients psilocin or psilocybin with erinacines or hericenones enhanced with niacin |
-
2019
- 2019-01-30 TW TW108103637A patent/TWI771561B/zh active
-
2020
- 2020-01-23 CN CN202010076480.5A patent/CN111494435A/zh active Pending
- 2020-01-29 JP JP2020012561A patent/JP6952812B2/ja active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN111494435A (zh) | 2020-08-07 |
| JP2020143043A (ja) | 2020-09-10 |
| TW202027756A (zh) | 2020-08-01 |
| JP6952812B2 (ja) | 2021-10-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Kim et al. | Interferon-γ promotes differentiation of neural progenitor cells via the JNK pathway | |
| Hong et al. | Therapeutic potential of induced neural stem cells for spinal cord injury | |
| Storch et al. | Long-term proliferation and dopaminergic differentiation of human mesencephalic neural precursor cells | |
| JP4023822B2 (ja) | ドーパミン作動性細胞のインビトロ誘導 | |
| Fariñas et al. | Characterization of neurotrophin and Trk receptor functions in developing sensory ganglia: direct NT-3 activation of TrkB neurons in vivo | |
| Monteiro et al. | Brain interference: revisiting the role of IFNγ in the central nervous system | |
| JP2008546385A (ja) | Ｃｎｓ疾患を治療するための単離された細胞および単離された細胞を含む集団 | |
| JP5632833B2 (ja) | 肝臓前駆細胞の馴化培地 | |
| EP2587265B1 (en) | Method for determination or evaluation of test substance | |
| CA2964927C (en) | Stable neural stem cells comprising an exogenous polynucleotide coding for a growth factor and methods of use thereof | |
| Zhang et al. | Synergistic effects of TGFβ2, WNT9a, and FGFR4 signals attenuate satellite cell differentiation during skeletal muscle development | |
| Guo et al. | Apomorphine induces trophic factors that support fetal rat mesencephalic dopaminergic neurons in cultures | |
| Hao et al. | Lipopolysaccharide and curcumin co-stimulation potentiates olfactory ensheathing cell phagocytosis via enhancing their activation | |
| Zheng et al. | The PI3K/Akt and ERK1/2 signaling pathways mediate the erythropoietin-modulated calcium influx in kainic acid-induced epilepsy | |
| Liu et al. | EGF signaling promotes the lineage conversion of astrocytes into oligodendrocytes | |
| Zhang et al. | Reducing host aldose reductase activity promotes neuronal differentiation of transplanted neural stem cells at spinal cord injury sites and facilitates locomotion recovery | |
| Moustardas et al. | MAPK pathways in ocular pathophysiology: potential therapeutic drugs and challenges | |
| US20120003186A1 (en) | Method for dedifferentiating adipose tissue stromal cells | |
| Wei et al. | Effects of engrafted neural stem cells derived from GFP transgenic mice in Parkinson's diseases rats | |
| He et al. | Peripheral nerve fibroblasts secrete neurotrophic factors to promote axon growth of motoneurons | |
| O’Toole et al. | Effect of olfactory ensheathing cells on reactive astrocytes in vitro | |
| Zhang et al. | JNK is necessary for oligodendrocyte precursor cell proliferation induced by the conditioned medium from B104 neuroblastoma cells | |
| TWI771561B (zh) | 猴頭菇菌絲體萃取物製備用於改善中樞神經系統髓鞘化之醫藥組合物之用途 | |
| Vandenberg et al. | Embryonal central neuroepithelial tumors: current concepts and future challenges | |
| Masjkur et al. | Neurovascular signals suggest a propagation mechanism for endogenous stem cell activation along blood vessels |