EFFECTS OF STEM BARK EXTRACT OF Alstonia boonei ON Plasmodium berghei berghei-INDUCED MALARIA IN MICE AND ON MITOCHONDRIAL PERMEABILITY TRANSITION PORE

Abstract

Mitochondrial Membrane Permeability Transition (MMPT) pore is a target for pharmacological intervention since the release of cytochrome c upon its opening is the point of no return for apoptosis. A number of antimalarial drugs have been shown to open the MMPT pore in folkloric medicine, the Stem Bark Extract of Alstonia boonei (SBEAB) is known to be useful in feverish conditions. However, there is paucity of information on whether SBEAB interferes with MMPT pore or not. In this study, the effect of SBEAB on Plasmodium berghei berghei-induced malaria in mice and on MMPT pore opening were investigated. The following fractions were partitioned from the Methanol Extract (ME) of SBEAB: n-hexane, Chloroform, Ethylacetate and Aqueous. The CF was subjected to Vacuum Liquid Chromatography (VLC) to give fractions: VLCF1, VLCF2, VLCF3 and VLCF4. Sixty-five male Swiss albino mice (18-20g) were orally treated with corn oil (control), fractions (200 and 400 mg/kg), chloroquine (5 mg/kg) and sulfadoxine-pyrimethamine (5 mg/kg). Seven days post-treatment, the mice were challenged with infected erythrocytes and were further treated for 7 days after confirmation of parasitemia. The effects of all the fractions on parasitemia and clearance were determined by microscopy. The ME was subjected to Accelerated Gradient Chromatography (AGC), to obtain fraction AGCF which was further subjected to 1D and 2D nuclear magnetic resonance and infra red spectroscopy. Rat liver mitochondria were prepared by differential centrifugation while MMPT pore opening was measured spectrophotometrically. Cytochrome C release, mitochondria ATPase activity and 1, 1-Diphenyl-2-picryl hydrazyl (DPPH) scavenging activity were assayed by spectrophotometry. Data were analysed using descriptive statistics Student's t-test and Duncan Multiple Range test at p=0.05. In the control animals, the percentage clearance (%) for CQ and SP were 100 each by day 3. The ME, HF, CF, EF. and AF had significant antiplasmodial activity with clearance of 68.1, 63.1, 88.4, 50.0 and 60.3 respectively. The VLC fractions showed varying antispasmodic activity with the highest %C of 88.7 by VLCF3 at day 7. In the control, the MMPT pore opening, was - 0.52±0.00 while the VLCF3 were -0.11±0.00, -0.06±0.00, -0.05±0.00 and -0.05 ±0.00 at 2.5, 5, 10 and 20ug/mL. respectively. The VLCF3 significantly inhibited the MMPT pore opening by 78.2, 88.6, 90.3 and 91.0% at 2.5, 5, 10 and 20ug/mL respectively. The MMPT pore opening for ME and AGCF were 7.86 (15 folds) at 20ug/ml and 5.24 (10 folds) at 10ug/mL respectively compared with the control. In the control, mitochondrial ATPase activity and cytochrome C release were 8.33±0.28 and 15.60±2.70 respectively while for the AGCF, they were 11.57±0.51 at 15ug/mL and 52.4±6.6 at 20ug/mL. Structural elucidation showed the presence of tetrahydro-4 -(E)-7-hydroxy-10-methoxy-6,14-dimethyl- 15-m-tolylpentadec- 13-enylpyran-2 -one (TMDTP), which was purified from Alstonia boonie, for the first lime. The TMDTP showed strong DPPH radical scavenging activity in vitro, confirming its antioxidant potency. The stem bark extract of Alstonia boonei exhibited antiplasmodial activity and induced the opening of the mitochondrial membrane permeability transition pore via cytochrome c release and mitochondria ATPase activity.