Methanol extract and fraction of Anchomanes difformis root tuber modulate liver mitochondrial membrane permeability transition pore opening in rats

Abstract

Objective: Extracts of Anchomanes difformis (AD) are used in folkloric medicine to treat several diseases and infections. However, their roles in mitochondrial permeability transition pore opening are not known.

Materials and methods: The viability of mitochondria isolated from Wistar rat liver used in this experiment, was assessed by monitoring their swelling amplitude in the absence of calcium and reversal of calcium-induced pore opening by spermine. The effects of methanol extract and fraction of A. difformis (MEAD and MFAD, respectively) on Mitochondrial Membrane Permeability Transition (MMPT) pore opening, ATPase activity, cytochrome c release and ferrous-induced lipid peroxidation were assessed spectrophotometrically. Phytochemical constituents of MEAD and MFAD were assessed using Gas Chromatography- Mass Spectrometry (GC-MS).

Results: The MEAD (10, 20, 40 and 80 μg/ ml) had no effect on MMPT pore opening in the absence of Ca²⁺, whereas MFAD at 80 μg/ml had a large amplitude pore opening effect. Both MEAD and MFAD reversed Ca²⁺‌‌-induced swelling with inhibition values of 18, 21, 24, 23% (for MEAD) and 41, 36, 35, and 26% (for MFAD) at 10, 20, 40 and 80 μg/ml, respectively. MFAD significantly enhanced F₁F₀ ATPase activity and caused cytochrome c release. Both MEAD and MFAD significantly inhibited ferrous-induced lipid peroxidation by 33.0, 64.0, 66, and 75% (for MEAD) and 24, 25, 30, and 45% (for MFAD), respectively. The GC-MS results revealed the presence of squalene as one of the major constituents of MEAD.

Conclusion: These findings suggest that MFAD can be used to induce cell death via mitochondrial permeability transition in isolated rat liver. Inhibition of lipid peroxidation by MEAD and MFAD showed that the pore opening effect of the extract and fraction was not mediated via peroxidation of mitochondrial membrane lipids.

Keywords: Anchomanes difformis; Cytochrome c; Lipid peroxidation; Mitochondrial permeability Transition pore opening; Phytoconstituents Mitochondrial ATPase.

Figure 1

Representative profile for the assessment of isolated rat liver mitochondrial permeability transition pore opening. Figure 1A shows the assessment of the mitochondria integrity in the absence of calcium, in the presence of calcium and reversal of calcium-induced mitochondrial membrane permeability transition pore opening by spermine. Figures 1B and 1C show the effect of varying concentrations of MEAD in the absence (B) and in the presence (C) of calcium on the mitochondrial membrane permeability transition pore opening. NTA: no triggering agent; TA: triggering agent

Figure 2

Representative profile showing the effects of MFAD on the mitochondrial permeability transition pore opening in the absence (A) and in the presence (B) of calcium. NTA: No triggering agent, TA: Triggering agent

Figure 3

Effect of MEAD and MFAD on F₁F₀ ATPase activity (Figure 3A) and cytochrome c release (Figure 3B) from rat liver mitochondria at physiological pH (7.4) (*^{, #} p<0.05, **^{, ##} p<0.01, ***^{, ###} p<0.001 all vs control (*) and DNP (^#)). In Figure 3B, there is a significant increase in the level of cytochrome c release at all concentrations of MFAD used when compared with the intact mitochondria. Bar represented the mean±SD. (n=4). (*p<0.05, ** p<0.01, *** p<0.001 all vs intact mitochondria)

Figure 4

Effect of varying concentrations of MEAD and MFAD on Fe²⁺-induced lipid peroxidation in normal rat liver mitochondria. Inhibition of lipid peroxidation by MEAD were statistically significant when 20, 40 and 80 µg/ml of MEAD were compared with corresponding concentrations of MFAD. Bar represented the mean±SD. (n=4). (***p<0.001)