Toxicity of Carlina Oxide-A Natural Polyacetylene from the Carlina acaulis Roots-In Vitro and In Vivo Study

Abstract

There are several reports indicating that the roots of the Carlina acaulis L. used to be commonly applied as a treatment measure in skin diseases and as an antiparasitic agent, starting from antiquity to the 19th century; however, nowadays, it has lost its importance. Currently, numerous studies are being conducted assessing the possibility of reintroducing C. acaulis-derived extracts to phytotherapy. Determining the safety profile of the main constituents of the plant material is crucial for achieving this goal. Here, we aimed to determine the toxicity profile of carlina oxide, one of the most abundant components of the C. acaulis root extract. We obtained the carlina oxide by distillation of C. acaulis roots in the Deryng apparatus. The purity of the standard was evaluated using GC-MS, and the identity was confirmed by IR, Raman, and NMR spectroscopy. In vitro cytotoxicity was assessed using a panel of human cell lines of skin origin, including BJ normal fibroblasts and UACC-903, UACC-647, and C32 melanoma cells. This was accompanied by an in vivo zebrafish acute toxicity test (ZFET). In vitro studies showed a toxic effect of carlina oxide, as demonstrated by an induction of apoptosis and necrosis in both normal and melanoma cells. Decreased expression of AKT kinase and extracellular signal-regulated kinase 1/2 (ERK1/2) was noted in the UACC-647 melanoma cell line. It was also observed that carlina oxide modified the expression of programmed cell death-ligand 1 (PD-L1) in tested cell lines. Carlina oxide exhibited high in vivo toxicity, with LC₅₀ = 10.13 µg/mL upon the 96 h of exposure in the ZFET test. Here, we demonstrate that carlina oxide displays toxic effects to cells in culture and to living organisms. The data indicate that C. acaulis-based extracts considered for therapeutic use should be completely deprived of carlina oxide.

Keywords: PD-L1; carlina oxide; cytotoxicity; folk medicine; phytotherapy; zebra fish.

Figure 1

Carlina oxide elicits cell-line-specific toxicity. Percentages of apoptotic, late apoptotic, and necrotic cells upon treatment with increasing concentrations of carlina oxide. Statistical analysis: two-way ANOVA with Dunnett’s post-hoc test; *, p < 0.05; **, p < 0.01; ***, p < 0.001. Data originates from three independent experiments.

Figure 2

Carlina oxide affects the expression of key signaling nodes in UACC-647 cells. (A) Representative Western blots obtained based on UACC-647 cells subjected to increasing concentrations of carlina oxide (3.125, 12.5, and 50 µg/mL) for 24 h. (B) Densitometric analysis of the expression of eukaryotic elongation factor 2 (eEF2), AKT, extracellular signal-regulated kinase 1/2 (ERK1/2), and proliferating cell nuclear antigen (PCNA). Statistical analysis: one-way ANOVA with Dunnett’s post hoc test; ***, p < 0.001; n/s, not significant. Data originated from four independent experiments.

Figure 3

Carlina oxide affects PD-L1 expression. (A) Expression of PD-L1 was studied in BJ fibroblasts and UACC-903, UACC-647, and C32 melanoma cells. The cells were exposed to a range of carlina oxide concentrations (3.125–50 µg/mL) and PD-L1 expression was assessed by qPCR. There was no detectable expression of PD-L1 in C32 cells. One-way ANOVA was conducted with Dunnett’s post hoc test; ***, p < 0.001; n/s, not significant. (B) Expression of miRNA previously linked with PD-L1 expression was studied in the same set of cell lines by qPCR. Obtained expression values (fold-change) are depicted as heatmap. Data originate from three independent experiments.

Figure 4

Carlina oxide is toxic to zebrafish embryos. (A) Mortality of zebrafish treated with carlina oxide (3.125–25 μg/mL) at the 96 h of the ZFET test. (B) Effects of carlina oxide on the heart rate of larval zebrafish at the 96 hpf of the ZFET test. Data represent mean ± SD, n = 10, *, p < 0.05 vs. negative control (E3), Dunnett’s test. (C) Teratogenic effects of carlina oxide on zebrafish larvae at the 96 h of the ZFET test (top panel: negative control – E3 medium; bottom panel: carlina oxide, 25 μg/mL). CFM, craniofacial malformation; YSO, yolk sac oedema; ST: shortened tail.