The Studies On The Chemical Constituents And Their Anti-cancer Activity From The Fruit Of Garcinia Subelliptica

OBJECTIVE: Garcina,the major genus within the family Guttiferae,is a prolific source of polycyclic polyprenylated acylphloroglucinols(PPAPs)and xanthones with diversity structures and biological activities.In order to elucidate the chemical constituents and biological activities,our group has been engaged in the systematic research for the chemical constituents and anti-cancer activity from the plant of the genera Garcinia distributed in China.As a continuous study for structurally diverse and bioactive PPAPs-type metabolites from the plant of the genera Garcinia,the investigation on the chemical constituents and anti-cancer activity of Garcinia subelliptica were carried out.METHODS: The chemical constituents from the fruit of the Garcinia subelliptica was systematically studied by means of many different chromatography methods,and the structures of these compounds were determined by comprehensive spectroscopic methods and single-crystal X-ray diffraction technique.MTT assay,flow cytometry and western blot were used to preliminarily evaluate the anti-cancer activity in vitro,from three aspects of cytotoxic activity,autophagy regulation and metastasis inhibition.RESULTS: Garsubelone A-Z(1-23,36-38)and garsubelone AA-AI(36-41,46-51),in total 35 new compounds and 24 known compounds(24-35,42-45,52-59),were isolated from the fruit of Garcinia subelliptica.Their plane structures were elucidated on the basis of the interpretation of NMR and MS spectroscopic data.The known compounds scrobiculaton A(30),scrobiculaton B(31),garcimultiflorone K(52),14-deoxygarcinol(53)and 18-hydroxy-7-epi-clusianone(57)were first isolated from the title plant.The absolute configurations of 1,10 and 36 were determined by X-ray diffraction technique.Structurally,the type-A PPAPs were mainly isolated from the seeds and seed coat,while the type-B PPAPs were isolated from the pericarp.Those compounds shared a common 8,8-dimethyl-bicyclo[3.3.1]nonane-2,4,9-trione core,and the side chains were substituted by isoprenyl group or geranyl.Isobutyryl,2-methylpentanoyl,benzoyl and hydroxylated benzoyl were as the acyl group.Nine new dimeric PPAPs featuring a complicated 6/6/6/6/6/6/6 heptacyclic architecture(1-9)were first isolated,those compounds was constructed by the plausible monomeric precursor,garsubelone B,K(10,11)and secohyperforin,via a key Diels-Alder cycloaddition to form the unique 2-oxabicyclo[3.3.1]nonane core,the absolute configurations of 1 and 10 were determined by X-ray diffraction data.In addition,four kinds of characteristic skeletons were characterized from the extracts: bicyclo [3.2.1]octone-2.4.8-trione core(36,47),cis-bicyclo[4.3.0]nonane core(33,44),1-9 seco BPAPs(51)and adamantane PPAPs(59).The anti-cancer activity of these isolates were also evaluated.Compounds 10,11,13,14,15,26,36 and 37 exhibited cytotoxic activities against four cancer cell lines He La,A549,MDA-MB-231 and Hep G2.Compounds 14,15,36,38 and 43 regulated mitophagy by promoting the accumulation of YFP-parkin.In particular,compounds 14 and 15 induced mitophagy by increasing the expression of PINK-1 and promoting the degradation of Tim23.Compounds 14,15,38 and 43 regulated autophagy by promoting the aggregation of GFP-LC 3.Compounds 14 and 15 induced autophagy by increasing the expression of LC3 II and reducing the expression of P62.Compound 37 induced apoptosis by increasing Sub-G1 and activating for Caspases 3 and 9.Compounds 11,13,14,15,26,36 and 37 exhibited metastasis inhibition against A549 in wound healing and transwell migration assay.Inaddition,compounds 36 and 37 inhibited metastasis by down-regulating the expression of metastasis related proteins of p-AKT/EMT/Cofilin singal pathway.CONCLUSION: Compounds 1-59,35 new compounds and 24 known ones,especially nine unprecedented dimeric PPAPs featuring with complicated 6/6/6/6/6/6/6 heptacyclic architecture(1-9),were isolated from the extracts of the fruit of Garcinia subelliptica.Plausible biogenetic pathways of dimeric PPAPs were proposed.The structural diversity of PPAPs was enriched by the identification of the dimeric PPAPs,type-A and type-B PPAPs,1,2-seco BPAPs,adamantane PPAPs,the change and difference of side chains.Compounds 10,11,13,14,15,26,36 and 37 exhibited cytotoxic activities and inhibition of metastasis.Compounds 37 induced apoptosis and inhibited metastasis by down-regulating the expression of related proteins.Compounds 14 and 15 induced mitophagy and autophagy by regulating the expression of related proteins.Compounds10,11,13,14,15,26,36,37 and 38 have potential anti-cancer activity.