Studies On Chemical Constituents,pharmacological Activities And Transcriptome Sequencing Of Trillium Tschonoskii

Trillium tschonoskii Maxim.(Liliaceae)is a herbaceous plant distributed at an altitude of 1600-3200 m in Qinba Moutains of China,locally known as 'Yan Ling Cao'.Its dried roots and rhizomes were used as a folk medicine for the treatment of neurasthenia,headache,cancer,and ameliorating pains,and various inflammatory diseases,such as sore,ulcer,fever and rheumatic pain.By means of various chromatographic methods such as silica gel,Sephadex LH-20,opening ODS column chromatography,and semi-preparative HPLC,21 compounds were isolated from the 70%ethanol extract of the roots and rhizomes of T.tschonoskii.Their structures were elucidated on the basis of spectroscopic data and physico-chemical methods.The compounds were comprised by 15 steroidal saponins,1 stilbene trimers,1 alkaloids,1 oligosaccharide esters and 1 sterol.Among them,compounds 1-4 were new,and named as furotrilliumoside(1),dioscin 6'-linoleate(2),trilliumoside(3),and 3a,3b,3c-tri-O-methylcyperusphenol D(12).The known compounds were identified as chonglouoside ?(5),pennogenin-3-O-?-L-rhamnopyranosyl(1?4)-[?-L-rhamnopyranosyl(1?2)]-?-glucopyranoside(6),dioscin(7),trikamsteroside C(8),trillenoside A(9),parispseudoside C(10),pseudoproto Pb(11),pseudoprotodioscin(12),pennogenin-3-O-?-L-rhamnopyranosyl-(1?4)-?-D-glucopyranoside(13),prosapogenin B(14),prosapogenin A(15),diosgenin(16),20-hydroxyecdysone(17),neoechinulin A(18)and(19)helonioside A.Among them,10-12 and 18 were obtained from the Trillium genus for the first time.Compounds 8,13 and 14 were obtained from the T.tschonoskii for the first time.The isolates were evaluated for their anti-inflammatory,and anti-tumor activities.The n-BuOH extract of the rhizomes of T.tschonoskii showed better inhibition of the dimethylbenzene induced auricle oedema in mice.The isolated compounds were further tested for their inhibitory activities on LPS-induced NO production in RAW264.7 cell by the Griess reaction.Among them,compound 1(FT)showed significant inhibitory effect with an IC50 value of 17.78 ?M,which was comparable to the positive control minocyline.FT could reduce LPS-induced expression of inducible iNOS and then result in the decrement of NO production.More meaningful,FT also down-regulated the expression of COX-2 and decreased the expressions of pro-inflammatory cytokines,TNF-?,IL-6 and IL-1?,both in the gene and protein levels.In mechanism study,FT blocked the LPS-induced upregulation of phosphorylated PI3K/Akt.Furthermore,FT inhibited the translocation of NF-?B phosphorylation and degradation and also suppressed the MAPKs signaling pathway.In addition,FT upregulated HO-1 expression via nuclear translocation of Nrf2.MTT results suggest that Compound 5 has stronger cytotoxicity and exhibits potent inhibitory activity against MCF-7,HCT-116,DU-145,SGC-7901,MCF-7/ADR and K562/ADR cell lines.Compound 5 induces cell autophagy and apoptosis via caspase-dependent mitochondrial pathway and the Akt/MAPKs pathway in K562/ADR leukemic cells.Compound 5 reverses multidrug resistance of K562/ADR leukemia cells by downregulating MDR1,MRP 1 gene and p-gp,ABCG2 protein expressions.Transcriptome of roots and rhizomes of T.tschonoskii was sequenced by high throughput sequencing techniques.32.72 M reads were achieved,and after de nove assemble,totally 210583 unigene sequences were obtained with average length of 502 bp.Bioinformatic analysis of Nr,GO,KOG,KEGG was carried out and we found a rich source of catalytic genes related with terpenoids,steroids and flavonoids biosynthesis.These information provided important foundation for the understanding of steroidal saponins biosynthesis.