Studies On The Antitumor Activity Of The Compounds Derived From Sinomenium Acutum (Thunb.) Rehd.et Wils.and The Secondary Production Of The Endophytic Fungus Talaromyces Amestolkiae CS-O-1 From Tripterygium Wilfordii Hook.F.

Glioblastoma?GBM?is the most common and lethal primary malignant tumor in human central nervous system,current therapies depend on surgical resection,chemotherapy and radiotherapy.The poor prognosis drives us to discover more potential natural products.Cannabisins is a lignanamide with different effects on different cancer cells,but its effects on GBM cells are still unclear.In this study,cannabisin G and cannabisin D were isolated from dried stem of Sinomenium acutum?Thunb.?Rehd.et Wils.by solvent extraction and various chromatographic methods,they were characterized by¹H-NMR and ¹³C-NMR.The human glioblastoma U87 and U251 cells were used to investigate their bioactivities.CCK-8 assay revealed that both of them significantly inhibited cell viability in a concentration dependent manner and determined EC50.The cell migration determined by transwell migration assay was also remarkably blocked by cannabisins.Apoptotic changes in nuclear morphology were observed after treatment with cannabisins by DAPI staining.Cell cycle and apoptosis changes before and after cannabisin D treatment were detected by flow cytometry.To further study the effect of cannabisin D on the cell cycle of GBM cells,cyclin protein was detected by Western blotting,cyclin and CDK gene transcription changes after treatment with cannabisin D were detected by real-time PCR.In order to explore its underlying mechanism,the phosphorylation of MAPKs in cell proteins after cannabisins treatment were detected by Western blotting,and it was found that activation of MAPKs is related to the inhibitory effect on GBM cells.In conclusion,cannabisin G and D were isolated from the Sinomenium acutum?Thunb.?Rehd.et Wils.for the first time,cannabisin G found to induce apoptosis in GBM cells partly through the activation of MAPKs,similarly,the inhibition of cannabisin D on human glioblastoma cell proliferation and migration is also related to the MAPKs pathwayNowadays,the research on microbial metabolites has become more and more extensive,and microbial-derived metabolites have become an important source of new active natural products.This research inherited the previous research on Tripterygium wilfordii Hook.f.endophytic fungi in this laboratory.Endophytic fungi from different parts of the medicinal plant Tripterygium wilfordii were isolated,purified,chemically screened.After screening,the metabolites of endophytic fungi CS-O-1 was found very abundant and may have immunosuppressive active metabolites.Therefore,we fermented the strain CS-O-1 to obtain a novel active lead compound.In this study,the species of the fungus CS-O-1 was identified as Talaromyces amestolkiae.Talaromyces amestolkiae CS-O-1 was fermented by 25 L,and the mycelium and filtrate were separated by filtration.The mycelia were extracted and partially separated.Fourteen compounds were separated by various methods,such as TLC,silica gel column chromatography,Sephadex LH-20 column chromatography,ODS reversed-phase column chromatography,and HPLC.Seven compounds were identified by MS and NMR,including a new compound named Chrodrimanin T?1?,and four known alkaloids nicotinamide?2?,penipyridones D?3?,penipyridones A?4?,3–benzylidene-8,8a–dihydroxy–2–methyl–hexahydro–pyrrolo[1,2-a]pyrazine–1,4-dione?5?,and two known esters aspergillumarin A?6?,butyl–isobutyl-phthalate?7?.