Cytotoxic Acetogenins from the Roots of Annona purpurea

Abstract

Annona purpurea, known in Mexico as "cabeza de negro" or "ilama", belongs to the Annonaceae family. Its roots are employed in folk medicine in several regions of Mexico. Taking that information into account, a chemical and biological analysis of the components present in the roots of this species was proposed. Our results demonstrated that the dichloromethane (DCM) extract was exclusively constituted by a mixture of five new acetogenins named annopurpuricins A-E (1-5). These compounds have an aliphatic chain of 37 carbons with a terminal α,β unsaturated γ-lactone. Compounds 1 and 2 belong to the adjacent bis-THF (tetrahydrofuran) α-monohydroxylated type, while compounds 3 and 4 belong to the adjacent bis-THF α,α'-dihydroxylated type; only compound 5 possesses a bis-epoxide system. Complete structure analysis was carried out by spectroscopy and chemical methods. All compounds were evaluated for their antiproliferative activity on three human tumor cell lines (MSTO-211H, HeLa and HepG2). Compounds 1-4 inhibited significantly the growth of HeLa and HepG2 cells, showing GI₅₀ values in the low/subnanomolar range, while 5 was completely ineffective under the tested conditions. The investigation of the mechanism of action responsible for cytotoxicity revealed for the most interesting compound 1 the ability to block the complex I activity on isolated rat liver mitochondria (RLM).

Keywords: Annona purpurea; Annonaceae; acetogenins; annopurpuricins; cytotoxicity; mitochondria.

Figure 1

Diagnostic ions in the EI mass spectrum of 1 (A) and 2 (B). R = H (natural compound) and R = TMSi [trimethylsilyl derivative (m/z 90)]. * Peak not observed.

Figure 2

Diagnostic ions in the EI mass spectrum of 3 (C), 4 (D), 5 (E). R = H (natural compound) and R = TMSi (trimethylsilyl derivative (m/z 90)). * Peak not observed.

Figure 3

Molecular model of annopurpuricin A (1).

Figure 4

Effects of compound 1 on mitochondrial ΔΨ. (A,B): RLM (1 mg/mL) are incubated in a medium containing succ (5 mM) and rot (1.25 µM). (C,D): RLM (1 mg/mL) are incubated in a medium containing glu (5 mM) and mal (1 mM). Test compound was added at indicated concentrations. When present 10 mM asc, 100 μM TMPD, 1.25 μM rot,1 μM ant A. Dotted lines represent the behavior of mitochondrial ΔΨ without the addition indicated by the arrow.

Figure 5

Effects of compound 1 on RCI and ADP/O ratio. (A,B): RLM (1 mg/mL) were incubated in a medium containing succ (5 mM) and rot (1.25 µM). (C–E): RLM (1 mg/mL) were incubated in a medium containing glu (5 mM) and mal (1 mM). Test compound was added at indicated concentrations. ADP concentration was 0.2 mM.

Figure 6

Effects of compound 1 on oxygen consumption in the absence or in the presence of the inhibitor of phosphorylation FCCP. (A,B): RLM (1 mg/mL) were incubated in a medium containing succ (5 mM) and rot (1.25 µM). (C,D): RLM (1 mg/mL) were incubated in a medium containing glut (5 mM) and mal (1 mM). Test compound was added at indicated concentration. FCCP concentration was 0.1 μg/mg protein.

Figure 7

Effects of compound 1 on mitochondrial swelling. (A): RLM (1 mg/mL) were incubated in a medium containing succ (5 mM) and rot (1.25 µM). (B,C): RLM (1 mg/mL) were incubated in a medium containing glu (5 mM) and mal (1 mM). Test compound was added at 1 μM (A,C) or at indicated concentrations (B). Ca²⁺ was present or added at 20 μM. Dotted line represents the behavior of mitochondrial swelling without the addition indicated by the arrow.

Figure 8

Effect of 1 on (A) mitochondrial ΔΨ of cells stained with JC-1 or (B) cell death mechanism. Dot plots from the flow cytometry analysis performed on HeLa cells incubated for 28 h in the absence (control) or in the presence of 5 nM compound 1. A representative experiment of at least three replicates is reported.