MODULATION OF MITOCHONDRIAL – MEDIATED APOPTOSIS BY SOLVENT FRACTIONS OF Daniellia oliveri (ROLFE) STEM BARK

Abstract

The Mitochondrial Permeability Transition (mPT) pore opening is a calcium-dependent process resulting from an increased permeability of the mitochondrial membrane. This mPT is a major factor which promotes apoptosis. Increasing mPT has evolved as a target for the treatment of tumours. Daniellia oliveri (DO) is a medicinal plant used in folkloric management of tumours in Africa. Compared to other tissues, liver cells have the highest concentration of mitochondria. Therefore, this study was carried out to investigate the effects of DO on mPT in rat livers.

The DO stem bark was collected, authenticated at Department of Botany, University of Ibadan (UIH: 22383), air dried, pulverized and extracted with ethanol for 72 hours. The filtrate gave ethanol extract (EEDO) that was partitioned successively with chloroform (CFDO), ethyl acetate (EAFDO) and ethanol (EFDO) to obtain three fractions of DO. An in vitro experiment was performed on liver mitochondria from fifteen male rats (110.0±1.8 g) using 60-300 µg/mL of EEDO and its fractions. Twenty-four male rats (90.0±0.5 g) were divided into four groups (n=6) and treated intraperitoneally for fourteen days thus; control (distilled water), 25, 50 and 100 mg/kg EFDO. After overnight fasting, rats were sacrificed and liver mitochondria were isolated by differential centrifugation (same as for in vitro). The mPT, mitochondrial ATPase (mATPase) and nuclear DNA (nDNA) fragmentation were assessed using spectrophotometry. Expressions of Bcl-2, Bax, p53 proteins and Cytochrome C Release (CCR) were determined using immunohistochemistry. Caspase 3 (C3) and Caspase 9 (C9) activities were determined by standard method using ELISA technique. The GC-MS was used to identify the compounds in EFDO. Data were analysed using descriptive statistics and ANOVA at α0.05.

The mPT was increased by 60, 120, 180, 240 and 300 µg/mL EEDO (0.03, 0.09, 0.20, 0.40, and 0.65 folds) and EFDO (0.04, 0.10, 0.35, 0.68, 0.75 fold), respectively. Graded concentrations (0.75, 2.25, 3.75, 4.25 and 6.75 µg/mL) of EFDO (3.2±0.2, 4.0±0.1, 4.5±0.3, 5.1±0.2, 5.9±0.1 µmpi/mg protein), respectively, CFDO (2.7±0.4, 3.0±0.2, 3.7±0.3, 4.1±0.3, 4.5±0.4 µmpi/mg protein) and EAFDO (2.9±0.1, 3.5±0.3, 4.3±0.2, 4.8±0.4, 5.1±0.1 µmpi/mg protein) enhanced mATPase activities relative to control (2.4±0.2 µmpi/mg protein), respectively. These five concentrations also increased CCR; EEDO (0.7±0.04, 0.8±0.03, 0.9±0.04, 1.1±0.06, 1.5±0.04 nmol/mg protein), EFDO (0.6±0.03, 0.7±0.03, 0.8±0.06, 0.9±0.02, 1.0±0.04 nmol/mg protein)

and CFDO (0.6±0.05, 0.7±0.02, 0.8±0.04, 0.9±0.03, 1.1±0.02 nmol/mg protein) relative to control (0.5±0.01 nmol/mg protein). The EFDO at 25, 50 and 100 mg/kg increased mPT (0.5, 0.6 and 0.7 folds) and mATPase activity (5.9±0.2, 7.0±0.4, 7.7±0.2 against 3.5±0.3 µmpi/mg protein). The Bax (35.6, 200.2 and 330.8%), p53 expression (91.3, 100.2 and 204.4%), CCR (50.2, 100.7 and 300.3%) and nDNA fragmentation (20.8, 55.2 and 115.6%) increased, while Bcl-2 (5.5, 34.5 and 56.4%) decreased compared to control. The C3 (15.8, 68.4 and 88.2%) and C9 (87.5, 150.5 and 180.4%) activities were increased compared to control. The GC-MS revealed the presence of oleic (54%) and palmitic acids (39%) in EFDO.

Daniellia oliveri increased apoptosis via mitochondrial membrane permeability transition pore opening. It also enhanced mitochondrial ATPase activity.