Chemical Constituents of Eupatorium japonicum and Anti-Inflammatory, Cytotoxic, and Apoptotic Activities of Eupatoriopicrin on Cancer Stem Cells

Abstract

Eupatorium japonicum Thunb. of the plant family Asteraceae is a popular traditional herb in Vietnam. However, its chemical constituents as well as bioactive principles have not been investigated yet. We investigated the phytochemistry of E. japonicum in Vietnam and isolated seventeen compounds (1-17) including phytosterols, terpenoids, phenolic acids, flavonoids, fatty alcohols, and fatty acids. They were structurally determined by MS and NMR analysis. Except for compounds 6 and 12, all the other compounds were identified for the first time from E. japonicum. Since many sesquiterpene lactones with α-methylene γ-lactone ring are reported as anti-inflammatory and anticancer agents, eupatoriopicrin (10), 1-hydroxy-8-(4,5-dihydroxytigloyloxy)eudesma-4(15),11(13)-dien-6,12-olide (11) were selected among the isolates for biological assays. Compound 10 was identified as the main bioactive sesquiterpene lactone of E. japonicum showing its potent anti-inflammatory and cytotoxic activity through inhibiting NO production and the growth of HepG2 and MCF-7 human cancer cell lines. For the first time, eupatoriopicrin (10) was demonstrated to strongly inhibit NTERA-2 human cancer stem cell (CSC) line in vitro. It is noticeable that the cytotoxicity of eupatoriopicrin against NTERA-2 cells is mediated by its apoptosis-inducing capability of 10 as demonstrated by the results of Hoechst 33342 staining, flow cytometry apoptosis analysis, and caspase-3 activity assays. The biological activities of the main bioactive constituents 1-7, 10, 12, and 15 supported the reported anti-inflammatory and anticancer properties of extracts from E. japonicum.

Figure 1

Structures of compounds 1–17.

Figure 2

Effectiveness of compounds 10 and 11 on different cell lines, including CSCs. Cultured cells (1 × 10⁴ cells/well) were treated with different concentrations of compounds ranging from 0.8 µg/mL to 100 µg/mL. DMSO 1% served as the negative control. Each value represents the mean ± SEM.

Figure 3

Nuclear condensation and fragmentation effects of compound 10 on NTERA cells at different concentrations as 1 µg/mL, 2 µg/mL, and 4 µg/mL using Hoechst 33342 staining. The cells were at 48 h of incubation and observed with Zeiss Vert A1 inverted microscope (100x).

Figure 4

Flow cytometry analysis of apoptosis-inducing activities of compound 10 at different concentrations ranging as 1 µg/mL, 2 µg/mL, and 4 µg/mL on NTERA-2 cells after 48 h incubation, using NovoCyte flow cytometry system and NovoExpress software (ACEA Bioscience, Inc.).

Figure 5

Caspase-3 inducible effects of compound 10 at different concentrations ranging from 0.5 µg/mL to 4 µg/mL on NTERA-2 cells after 48 h incubation. Diluted solution DMSO 1% served as the negative control. Each value represents the mean ± SEM. ^∗∗p < 0.01 and ^∗p < 0.05 compared to the negative control.