| The chemical constituents and biological activity of Juniperus oblonga M.Bieb.and Juniperus rigida Sieb.et Zucc.were investigated,and compounds have been isolated with anti-neuroblastoma activity,organic anion transporter 3（OAT3）inhibitory activity and cytotoxic activity.Juniperus oblonga is not found in China and samples were collected in Azerbaijan for these experiments.Juniperus rigida is widely distributed in China.As the initiator and presenter of“the Belt and Road”,the phytochemical investigation of Juniperus rigida not only provides a new prospect for pharmaceutical development,it also improves the utilization of resource research in China.The main research contents and results of this thesis are as follows:（1）Study on the inhibitory effect of biflavones on organic anion transporter 3By using macroporous adsorption resin,HPLC and other separation methods,three bioflavonoids,amentoflavone,cupressuflavone and podocarpusflavone A,were isolated from the dichloromethane and n-butanol soluble fractions of Juniperus oblonga which exhibited strong inhibition of OAT3.Amentoflavone showed the strongest inhibitory activity with an IC50 of 2.0μM.These biflavonoids are at least an order of magnitude more potent than their corresponding monomer,apigenin.This result provides a new point for drug development and synthesis.In vivo studies also suggested that amentoflavone have potential clinical used as OAT3 inhibitor.（2）Bioactive compounds from Juniperus oblonga inhibit neuroblastomaUsing silica gel column chromatography,HPLC and other separation methods,four compounds were isolated from the dichloromethane extract of Juniperus oblonga and identified as（-）-deoxypodophyllotoxin,（-）-matairesinol,（+）-isocupressic acid and（-）-isocupressic acid.The effects of these compounds on apoptosis and cell cycle progression were investigated using sulforhodamine B assay,mitochondrial permeability assay,cell calcium flux assay and Western blot.（-）-Deoxypodophyllotoxin,（-）-matairesinol and（+）-isocupressic acid exhibited strong inhibition for both neuroblastoma and high-risk neuroblastoma.Additionally,（-）-deoxypodophyllotoxin exhibited strongest inhibitory with IC50 of 4.27 n M,and posseses a different mechanism of action than other two compounds.These results may provide a new opportunity for clinical development of neuroblastoma drugs and lead to more effective treatment options for high-risk neuroblastoma patients.（3）Diverse compounds from dichloromethane extracts of Juniperus rigida and Juniperus oblonga and their cytotoxic activitiesThe dichloromethane extracts from Juniperus oblonga and Juniperus rigida were investigated by using TLC,HPLC and other separation methods leading to the isolation of one previously undescribed labdane diterpenoid,（4R,5S,9S,10R）-13-des-ethyl-13-oxolabda-8（17）,11E-dien-19-oic acid,together with five known labdane diterpenoids identified as 15-nor-14-oxolabda-8（17）,12Z-dien-19-oic acid,sandaracopimaric acid,lanceolatanoic acid,15-hydroxylabd-8（17）-en-19-oic acid and trans-communic acid.Eight abietane diterpenoids were identified as sugiol,6,12-dihydroxyabieta-5,8,11,13-tetraen-7-one,ferruginol,7α-methoxydeoxocryptojaponol,sempervirol,trilobinol,7-hydroxydeoxo-cryptojaponol and 7-oxodehydroabietinol.One sesquiterpene was identified as thujopsen-12-ol.Two lignans were identified as（-）-yatein and helioxanthin.A coumarin,umbelliferone,and a small phenolic compound,4-methoxy-3-（2-methylpropyl）-phenol were also isolated and identified.Sixteen compounds are first reported from Juniperus oblonga and Juniperus rigida,and ten compounds are here reported from the genus Juniperus for the first time.In addition,the isolated compounds were evaluated for their cytotoxic activities against three human tumor cell lines（Hep G2,MCF-7 and He La）.Sempervirol showed modest activity and 6,12-dihydroxyabieta-5,8,11,13-tetraen-7-one and thujopsen-12-ol exhibited moderate cytotoxicity against all three cell lines.The absence of cytotoxicity against the normal human liver cell line（L-02）may also have some importance.In summary,the isolation,structure elucidation and mechanism of bioactive compounds from Juniperus oblonga are primarily described,as well as an additional phytochemical investigation of Juniperus rigida which is a widely cultivated species in China.Some active compounds have been previously reported from this plant which may greatly improve the medicinal value of Juniperus rigida.The isolated compounds,including flavonoids,terpenoids,and lignans,enrich our understanding of the chemical diversity of Juniperus.These results may promote the utilization of the Juniperus genus in China for medical uses and as a highly promising natural product resource. |
