Carlina acaulis Exhibits Antioxidant Activity and Counteracts Aβ Toxicity in Caenorhabditis elegans

Abstract

Carlina acaulis is a medicinal plant that has shown antioxidant activity in in vitro studies, but to date no corresponding in vivo data is available. Therefore, in the present study the antioxidant activity and its impact in counteracting Aβ toxicity were studied in the Caenorhabditis elegans model. A dichloromethane extract of the roots of C. acaulis was prepared and characterised via gas-liquid-chromatography/mass-spectrometry (GLC-MS). The in vitro antioxidant activity was confirmed via 2,2-diphenyl-1-picrylhydracyl assay. The extract was further separated by thin layer chromatography into two fractions, one of which was a fraction of the dichloromethane extract of C. acaulis containing mostly Carlina oxide (CarOx). Different strains of C. elegans were employed to study the expression of hsp-16.2p::GFP as a marker for oxidative stress, delocalisation of the transcription factor DAF-16 as a possible mechanism of antioxidant activity, the effect of the drug under lethal oxidative stress, and the effect against beta-amyloid (Aβ) toxicity in a paralysis assay. The C. acaulis extract and CarOx showed high antioxidant activity (stress reduction by 47% and 64%, respectively) in C. elegans and could activate the transcription factor DAF-16 which directs the expression of anti-stress genes. In paralysis assay, only the total extract was significantly active, delaying paralysis by 1.6 h. In conclusion, in vivo antioxidant activity was shown for C. acaulis for the first time in the C. elegans model. The active antioxidant compound is Carlina oxide. This activity, however, is not sufficient to counteract Aβ toxicity. Other mechanisms and possibly other active compounds are involved in this effect.

Keywords: Caenorhabditis elegans; Carlina acaulis; Carlina oxide; antioxidant; beta-amyloid.

Figure 1

Total ion current of the GLC-MS measurements for the total dichloromethane extract of C. acaulis (A); fraction of the dichloromethane extract of C. acaulis containing mostly Carlina oxide (CarOx) (B); and the depleted extract (C).

Figure 2

Heat shock protein (HSP) expression assay in C. elegans. A–F: representative pictures of GFP fluorescence in worms treated with 0.5% methanol (A; negative control); 50 μg/mL total extract of C. acaulis (B); 25 μg/mL depleted extract (C); 25 μg/mL CarOx (D); 100 μg/mL (−)-epigallocatechin gallate (EGCG) (E; positive control); and 200 μg/mL ascorbic acid (F; positive control); (G) quantification of the fluorescence intensity for respective treatments. Pictures taken with BZ9000 from Keyence, scale bar = 100 μm. ** p < 0.01 compared to the negative control.

Figure 3

Results of the DAF-16 delocalisation assay in C. elegans. (A) A worm treated with 0.5% methanol (negative control); (B) a worm treated with 25 μg/mL CarOx. Pictures taken with BZ9000 from Keyence, scale bars = 100 μm; (C) quantification of the results. ** p < 0.01 compared to the negative control.

Figure 4

Survival assay in C. elegans. The worms were treated with indicated substances prior to subjecting them to lethal oxidative stress induced by 80 μM juglone. ** p < 0.01 compared to the negative control (methanol).

Figure 5

Paralysis curves for C. elegans expressing Aβ (CL4176) treated with dichloromethane extract of C. acaulis (Carlina), depleted extract, or CarOx in concentrations 25 μg/mL (A) and 50 μg/mL (B) with respective solvent controls.