Study On Chemical Constituents From Cannabis Sativa L.Subsp.Sativa And Their Effects On Neurodegenerative Diseases

In this thesis,the study on protective effects of Cannabis sativa L.subsp.sativa on neurodegenerative diseases was carried out with Aβ-42-induced spatial memory impairment in mice,quinolinic acid-induced learning and memory deficits in rat and MPP+-induced Parkinson’s disease(PD)model in SH-SY5 Y cells.The results showed that the 70%EtOH extract of the stems and leaves,the fruits of C.sativa could protect against Alzheimer’s disease(AD).And the EtOAc fraction of the stems and leaves of C.sativa is the effective part.The 70%EtOH extract of the stems and leaves of C.sativa could protect against PD,and the EtOAc fraction of the stems and leaves is the active site.An investigation was carried out on the EtOAc fraction of the 70%EtOH extract of the stems and leaves,and fruits of C.sativa,respectively.60 compounds were isolated through various chromatographic techniques,such as silica gel column chromatography(CC),Sephadex LH-20 CC,octadecylsilyl(ODS)CC and high performance liquid chromatography(HPLC).On the basis of physical chemical properties,spectroscopic analyses,calculated electronic circular dichroism(ECD)and Rh2(OCOCF3)4-induced CD method,their structures were identified.46 compounds were isolated from the stems and leaves of C.sativa,and their structures were identified as(9R,10S)-9-hydroxy-10-methoxy-⊿6a(10a)-tetrahydrocannabivarin(1*),(9R,10S)-9hydroxy-l 0-methoxy-⊿6a(10a)-tetrahydrocannabinol(2*),(6aS,9S,10R,10aR)-9,10-epoxy-10ahydroxy-tetrahydrocannabivarin(3*),(7Z,9Z)-cannabiphenolic acid A(4*),(8S,9Z)cannabiphenolic acid B(5*),cannabitriol-C3(6),10α-hydroxy-⊿9,13-hexahydrocannabinol(7),⊿910b-hydroxyl-tetrahydrocannabinol(8),⊿9-trans-tetrahydrocannabivarin(9),cannabivarin(10),cannabinol(11),cannabichromevarin(12),cannabichromene(13),cannabichromanone(14),acetyl cannabispirol(15),cannabispirol(16),canniprene(17),9,10-dihydro-2,3,5,6tetramethoxyphenanthrene-1,4-dione(18),⊿-oleanolic acid(19),erythrodiol(20),oleanolic acid(21),3β-hidroxyolean-12-en-11-one(22),maslinic acid(23),camaldulenic acid(24),3βhydroxyanticopalic acid(25),p-hydroxyenzaldehyde(26),salicylic acid(27),syringic acid(28),vanillin(29),4-hydroxy-3-methoxyacetophenone(30),3-hydroxy-4-methylacetophenone(31),3hydroxy-6-methylacetophenone(32),1-methoxy-3-indoleacetonitrile(33),sedanolide(34),senkyunolide(35),3-hydroxy-butylphthalide(36),coniferaldehyde(37),(E)-methyl phydroxycinnamate(38),(Z)-methyl p-hydroxycinnamate(39),ferulic acid(40),(+)monomethylpinoresinol(41),(-)-phillygenin(42),(-)-pinoresinol(43),(-)-epipinoresinol(44),oroxylin A(45),skullcapflavone Ⅱ(46),5 compounds(1-5)are new compounds,27 compounds(19-20,22-46)were reported from the Cannabis genus for the first time.14 compounds were isolated from the fruits of C.sativa,and their structures were identified as(1S,2R,3S,4R)-cannabisin P(47*),cannabisin A(48),cannabisin B(49),grossamide(50),N-pcoumaroyltyramine(51),N-trans-caffeoyltyramine(52),N-trans-caffeoyloctopamine(53),N-transferulyltyramine(54),cannabisin F(55),N1,N5,N10-tri-p-coumaroylspermidine(56),protocatechuic acid methyl ester(57),indolic acid(58),p-hydroxycinnamic acid(59),caffeic acid(60).Compound 47 is a new compound,5 compounds(53,56-57,59-60)were reported from the Cannabis genus for the first time.The neuroprotective effects of 13 compounds(1,9,11,16,20-21,23,26,38-40,42,46)from the stems and leaves of C.sativa and 9 compounds(48-55,60)from the fruits of C.sativa were investigated by H2O2-induced injury model in PC 12 cells.The results showed that compounds 26,38,39,40,52 and 55 showed protective effects.14 compounds(10-14,18-19,26,29,31,35,37-39)from the stems and leaves of C.sativa were estimated their protective action on MPP+-induced PD model in SH-SY5Y cells.The results showed that compounds 12,26 and 39 could alleviate MPP+induced cytotoxicity.Compound 39 was identified to bind HIPK2 with high affinity through molecular modeling and molecular dynamics(MD)simulations.The possible mechanism of 39 was studied through cell morphology,flow cytometry and western blot assay,indicating that 39 could attenuate MPP+-induced apoptosis in SH-SY5Y cells,but further research should be needed.