In Vitro and In Silico Anti-Glioblastoma Activity of Hydroalcoholic Extracts of Artemisia annua L. and Artemisia vulgaris L

Abstract

Glioblastoma, the most aggressive and challenging brain tumor, is a key focus in neuro-oncology due to its rapid growth and poor prognosis. The C6 glioma cell line is often used as a glioblastoma model due to its close simulation of human glioma characteristics, including rapid expansion and invasiveness. Alongside, herbal medicine, particularly Artemisia spp., is gaining attention for its anticancer potential, offering mechanisms like apoptosis induction, cell cycle arrest, and the inhibition of angiogenesis. In this study, we optimized extraction conditions of polyphenols from Artemisia annua L. and Artemisia vulgaris L. herbs and investigated their anticancer effects in silico and in vitro. Molecular docking of the main phenolic compounds of A. annua and A. vulgaris and potential target proteins, including programmed cell death (apoptosis) pathway proteins proapoptotic Bax (PDB ID 6EB6), anti-apoptotic Bcl-2 (PDB ID G5M), and the necroptosis pathway protein (PDB ID 7MON), mixed lineage kinase domain-like protein (MLKL), in complex with receptor-interacting serine/threonine-protein kinase 3 (RIPK3), revealed the high probability of their interactions, highlighting the possible influence of chlorogenic acid in modulating necroptosis processes. The cell viability of rat C6 glioma cell line was assessed using a nuclear fluorescent double-staining assay with Hoechst 33342 and propidium iodide. The extracts from A. annua and A. vulgaris have demonstrated anticancer activity in the glioblastoma model, with the synergistic effects of their combined compounds surpassing the efficacy of any single compound. Our results suggest the potential of these extracts as a basis for developing more effective glioblastoma treatments, emphasizing the importance of further research into their mechanisms of action and therapeutic applications.

Keywords: Artemisia annua L.; Artemisia vulgaris L.; C6 glioma cell line; chlorogenic acid; glioblastoma.

Figure 1

Plants of Artemisia annua L. (a) and Artemisia vulgaris L. (b).

Figure 2

Chemical structures of main phenolic compounds detected in Artemisia annua L. and Artemisia vulgaris L. herbal hydroalcoholic extracts. 1—apigenin, 2—luteolin, 3—neochlorogenic acid, 4—chlorogenic acid, 5—4-o-caffeoylquinic acid, 6—caffeic acid, and 7—isoquercitrin.

Figure 3

Yields of main polyphenolic compounds (low values (a), high values (b)) from herbal hydroalcoholic extracts of Artemisia annua L. and Artemisia vulgaris L. Data are presented as mean ± standard error (SEM), n = 4. * p < 0.05—statistically significant difference compared to corresponding Artemisia annua samples. The results were analyzed with a one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test.

Figure 4

Total amount of phenolic compounds in herbal hydroalcoholic extracts of Artemisia annua L. and Artemisia vulgaris L. in the presence and absence of the excipient L-glutathione (1%). Data are presented as mean ± SEM, n = 4. * p < 0.05—statistically significant difference compared to control without the excipient; # p < 0.05—statistically significant difference of A. vulgaris samples compared to A. annua samples. The results were analyzed with a one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test.

Figure 5

Docking of chlorogenic acid to MLKL/RIPK3 protein complex forms a necroptosis pathway. MLKL depicted in yellow, RIPK3 is depicted in red. Molecular docking studies were carried out using AutoDock Vina 4.05. All non-protein residues were removed, retaining pure protein structure for docking simulations. The structure presented has the lowest docking energy (−6.8 kcal/mol) and the highest number of hydrogen bonds (N18).

Figure 6

Effects of different concentrations of Artemisia annua L. extract without (a) and with the excipient—1% of L-glutathione (b) on the viability of C6 cells. C6 cells were treated with different concentrations of extract (5–50 µg/mL of phenolic compounds) for 24 h. Data are presented as means of percentage of the untreated control cells ± SEM (n = 5). * p < 0.05 versus control, ^# p < 0.05 versus extract without the excipient. The results were analyzed with one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test.

Figure 7

Effects of different concentrations of Artemisia annua L. extract without (a) and with the excipient—1% of L-glutathione (b) on viability of C6 cells. C6 cells were treated with different concentrations of extract (5–70 µg/mL of phenolic compounds) for 24 h. Data are presented as means of percentage of the untreated control cells ± SE (n = 5). * p < 0.05 versus control, ^# p < 0.05 versus extract without the excipient. The results were analyzed with one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test.

Figure 8

Effects of different concentrations of chlorogenic acid on viability of C6 cells. C6 cells were treated with different concentrations (5–70 µg/mL) of chlorogenic acid for 24 h. Data are presented as means of percentage of the untreated control cells ± SE (n = 5). * p < 0.05 versus control. The results were analyzed with one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test.

Figure 9

Effects of different concentrations of A. annua L. hydroalcoholic extract on viability of C6 cells. Cells were double-stained with Hoechst 33342 and PI, and the viability was assessed under fluorescence microscope. Original magnification ×20. Typical photographs of control cells (a) and after treatment with (b) 10 µg/mL phenolic compounds, (c) 20 µg/mL phenolic compounds (d) 50 µg/mL phenolic compounds of investigated extract. Hoechst 33342-positive cells, exhibiting blue fluorescence, were considered viable cells. PI-stained cells exhibiting red fluorescence were considered necrotic.