Cytotoxic Properties of Three Isolated Coumarin-hemiterpene Ether Derivatives from Artemisia armeniaca Lam

Abstract

Considering multiple reports on cytotoxic activity of the Artemisia genus and its phytochemicals, in the current study A. armeniaca Lam. and the three components isolated from the plant were subjected to cytotoxic studies. Analytical fractionation of A. armeniaca aerial parts for the first time was directed to the isolation of 7-hydroxy-8-(4-hydroxy-3-methylbutoxy) comarin (armenin), 8-hydroxy-7-(4-hydroxy-3-methylbutoxy) comarin (isoarmenin) and deoxylacarol. Cytotoxicity assessed with alamalBlue® assay and apoptosis was detected by PI staining and western blot analysis of Bax and PARP proteins. Extracts and all compounds exhibited cytotoxic activity against apoptosis-proficient HL-60 and apoptosis-resistant K562 cells, with the lowest cytotoxic activity on J774 cell line as non-malignant cell. Armenin as the most potent component decreased the viability of cell with IC50 of 22.5 and 71.1 µM for K562 and HL-60 cells respectively and selected for further mechanistic study. Armenin increased the sub-G1 peak in flow cytometry histogram of HL-60 and K562 treated cells and increase in the amount of Bax protein and the cleavage of PARP in comparison with the control after treatment for 48 h in K562 treated cells verified the apoptotic activity of the armenin. Taken together, according to the finding of this study armenin was introduced as a novel cytotoxic compound with apoptotic activity, which is encouraging for further mechanistic and clinical studies.

Keywords: Artemisia armeniaca; Asteraceae leukemic cell lines; apoptosis; coumarin-hemiterpene ether derivatives.

Figure 1

Partitioning scheme of A. armeniaca

Figure 2

The concentration-dependent effects of CH2Cl2 and MeOH extracts (0-200 μg/ml) and 7-hydroxy-8-(4-hydroxy-3-methylbutoxy) comarin (armenin), 8-hydroxy-7-(4-hydroxy-3-methylbutoxy) comarin (isoarmenin) and deoxylacarol on the viability of K562 and HL-60 cells. Among three compounds armenin exhibited high cytotoxic activity against apoptosis-proficient HL-60 and apoptosis-resistant K562 cells, with IC50 values ranging from 22.5 to 71.1 μM with much less cytotoxic effects on normal human lymphocytes. Paclitaxel 1 μM was used as positive control. Values were mean±SD of at least three independent experiments, each in triplicates. +P < 0.05, ++P < 0.01 and +++P < 0.001 compared to control.

Figure 3

Percentage of sub-G1 peak after PI staining and flow cytometry analysis of armenin (0.0, 25 and 50 µM) showing apoptosis induction in K562 and HL-60 cells. All experiments were done in triplicate. *P < 0.05 compared to control

Figure 4

Proteolytic cleavage of poly (ADP-ribose) polymerase (PARP) in K562 cells after 48 h exposure to armenin (0.0, 12.5 and 50 µM). β-Actin was used as a loading