Cytotoxic and natural killer cell stimulatory constituents of Phyllanthus songboiensis

Abstract

A dichapetalin-type triterpenoid and a dibenzylbutyrolactone-type lignan, together with five known lignans, a known aromatic diterpenoid, and a known acylated phytosterol, were isolated from the aerial parts of Phyllanthus songboiensis, collected in Vietnam. Their structures were determined by interpretation of the spectroscopic data, and the inhibitory activity toward HT-29 human colon cancer cells of all isolates was evaluated by a cytotoxicity assay. The known arylnaphthalene lignan, (+)-acutissimalignan A, was highly cytotoxic toward HT-29 cells, with an IC50 value of 19 nM, but this compound was inactive as a DNA topoisomerase IIα (topo IIα) poison. The known phytosterol, (-)-β-sitosterol-3-O-β-D-(6-O-palmitoyl)glucopyranoside, was found to stimulate natural killer (NK) cells at a concentration of 10μM in the presence of interleukin 12 (IL-12).

Keywords: Cytotoxicity; DNA topoisomerase IIα; Lignan; Natural killer cell; Phyllanthaceae; Phyllanthus songboiensis; Phytosterol.

Fig. 1

Chemical structures of compounds 1−5.

Fig. 2

¹H-¹H COSY and key HMBC correlations for 1 and 2 and selected NOESY correlations for 2.

Fig. 3

Molecular model and selected NOESY correlations for 1. Molecular model of 1 was obtained from Chem3D Pro 12.0 program, and the NOESY spectrum was measured on a Bruker DRX-800 MHz NMR spectrometer.

Fig. 4

Evaluation of (+)-acutissimalignan A (3) as a topo IIα poison. Topo II-DNA covalent complexes induced by 3 and etoposide were trapped by rapidly denaturing the complexed enzyme with sodium dodecyl sulfate (SDS), digesting the enzyme, and releasing the cleaved DNA as linear DNA. The formation of linear DNA was detected by separating the SDS-treated reaction products using ethidium bromide gel electrophoresis and quantified by accounting for the relationship between fluorescence and relative band intensity for open circular (OC), linear (LNR), supercoiled (SC), and RLX (relaxed) configurations of DNA.

Fig. 5

Production of IFN-γ by NK cells stimulated by IL-12 and 5. Human natural killer cells were treated by vehicle, IL-12 (10 ng/mL), or IL-12 (10 ng/mL) plus 5 (10 μM), for 24 h. Cells were harvested, and cell-free supernatants were collected. The level of IFN-γ protein in the collected cell-free supernatants was detected by an enzyme-linked immunosorbent assay (ELISA). The result showed that 5 stimulated NK cell to produce higher levels of IFN-γ in the presence of IL-12, compared to IL-12 alone (columns, mean values, n = 3; bars, SEM; * p ≤ 0.05 and ** p ≤ 0.01).