Novel steroidal saponin isolated from Trillium tschonoskii maxim. exhibits anti-oxidative effect via autophagy induction in cellular and Caenorhabditis elegans models.

Emerging evidences indicate the important roles of autophagy in anti-oxidative stress, which is closely associated with cancer, aging and neurodegeneration.In the current study, we aimed to identify autophagy inducers with potent anti-oxidative effect from traditional Chinese medicines (TCMs) in PC-12 cells and C. elegans.

The autophagy inducers were extensively screened in our herbal extracts library by using the stable RFP-GFP-LC3 U87 cells. The components with autophagic induction effect in Trillium tschonoskii Maxim. (TTM) was isolated and identified by using the autophagic activity-guided column chromatography and Pre-HPLC technologies, and MS and NMR spectroscopic analysis, respectively. The anti-oxidative effect of the isolated autophagy inducers was evaluated in H₂O₂-induced PC-12 cells and C. elegans models by measuring the viability of PC-12 cells and C. elegans, with quantitation on the ROS level in vitro and in vivo using H₂DCFDA probe.

The total ethanol extract of TTM was found to significantly increase the formation of GFP-LC3 puncta in stable RFP-GFP-LC3 U87 cells. One novel steroidal saponin 1-O-[2,3,4-tri-O-acetyl-α-L-rhamnopyranosyl-(1→2)-4-O-acetyl-α-L-arabinopyranosyl]-21-Deoxytrillenogenin, (Deoxytrillenoside CA, DTCA) and one known steroidal saponin 1-O-[2,3,4-tri-O-acetyl-α-L-rhamnopyranosyl-(1→2)-4-O-acetyl-α-L-arabinopyranosyl]-21-O-acetyl-epitrillenogenin (Epitrillenoside CA, ETCA) were isolated, identified and found to have novel autophagic effect. Both DTCA and ETCA could activate autophagy in PC-12 cells via the AMPK/mTOR/p70S6K signaling pathway in an Atg7-dependent. In addition, DTCA and ETCA could increase the cell viability and decrease the intracellular ROS level in H₂O₂-treated PC-12 cells and C. elegans, and the further study demonstrated that the induced autophagy contributes to their anti-oxidative effect.

Our current findings not only provide information on the discovery of novel autophagy activators from TTM, but also confirmed the anti-oxidative effect of the components from TTM both in vitro and in vivo.