© 2017 Journal of Basic and Clinical Pharmacy 221 ORIGINAL ARTICLE INTRODUCTION Mitochondria play an important role in cell death because of its prominent feature in apoptotic signal transduction and amplifcation. [1,2] Te complex role of mitochondria in apoptosis came into focus when biochemical studies identifed several mitochondrial proteins that are able to activate cell death directly via mitochondrial-mediated pathway. [3] Prior to apoptosis, mitochondria sufer specifc damages that result in release of cytochrome c, the sole water-soluble component of the electron transfer chain, and this can potentially halt the electron transfer leading to failure in maintaining the mitochondrial membrane potential that is needed in ATP synthesis. [4] It has been established that once released, cytochrome c binds to apoptotic protease activator-1 (APAF-1) in the presence of ATP or dATP and forms a complex that activates pro-caspase-3 and -7. [5] In cell-free systems, it has been demonstrated that mitochondria are necessary components of the cytosolic fraction to produce apoptotic features. [6] Loss of mitochondrial ATP synthesis has been reported in cells afer growth-factor deprivation. [7] In this apoptotic cells, cellular ATP content decreased when cytosolic ADP and inter- membrane creatinine phosphate concentration increase. Te MMPT is a phenomenon whereby the exquisitely controlled permeability of the inner membrane is disrupted and mitochondria become non- selectively permeable to solutes up to 1.5 kD. [8,9] Associated with this is the rapid equilibration of solutes across the mitochondrial membranes leading to depolarization of membrane potential, osmotic swelling, and release of potential apoptogenic actors including cytochrome c. [10] Tus, the MMPT-induced loss of mitochondrial function, release of cytochrome c, and the resultant ROS generation account for cell death. Te MMPT pore is thought to be a multi-protein complex which assembles at the sites of contact between the inner and outer mitochondrial membranes. [11] Te pore normally remains closed, but can open under conditions of cellular stress with dire consequences. When the MMPT pore opens, the permeability barrier of the inner membrane becomes disrupted, all small molecular weight solutes move freely across the membrane and as a result, they exert a colloidal osmotic pressure that causes mitochondria to swell and the unfolding of the cristae allows the matrix to expand without rupture of the inner membrane, the outer membrane will break and lead to the release of proteins in the inter-membrane space such as cytochrome c. Also, the inner membrane becomes freely permeable to protons. Tis uncouples oxidative phosphorylation, causing the proton-translocating ATPase to reverse direction and so actively hydrolyse ATP, rather than synthesize it. [12] Under such conditions, intracellular ATP concentrations rapidly decline, leading to the disruption of ionic and metabolic homeostasis and the activation of degradative enzymes such as phospholipases, nucleases, and proteases. Te key factors responsible for MMPT pore opening are: mitochondrial calcium overload (i.e., when mitochondrial matrix [Ca 2+ ] is greatly increased), presence of pro-oxidants, chemotherapeutic agents and proapoptotic Bcl-2 family members (e.g., Bax, Bak, and Bid). [13] Te pivotal role played by mitochondria in cell death has made them a vulnerable target for experimental and pharmacological intervention. [14] It has been suggested that certain bioactive agents in medicinal plants could elicit chemo-protective and therapeutic efects through the induction or inhibition of the opening of the MMPT. [15] A. boonei (De Wild) is a very large deciduous, tropical forest tree that belongs to the dogbane family, Apocynaceae which consists of about 50 species widely distributed in the continents of Africa, Asia and America. ABSTRACT Background: The Mitochondrial Membrane Permeability Transition (MMPT) pore is an important target for the development of cytotoxic drugs because the release of cytochrome c and the irreversible opening of the pore is a point of no return for apoptosis to take place. A. boonei is a perennial plant that is quite ubiquitous in rain forest and sub-Saharan regions and it is used in folklore medicine as an anti-malarial decoction. Objective: To assess the effects of Vacuum Liquid Chromatography (VLC) chloroform (100%) fraction purifed from the stem bark extract of A. boonei on MMPT pore of mitochondria isolated from livers of male rats in the presence and absence of added calcium ions. Materials and Methods: The in vitro effects of the VLC chloroform fraction on MMPT were assessed by monitoring the amplitude swelling spectrophotometrically as decreases in absorbance at 540 nm. Results: The MMPT pore was opened by six folds in the presence of calcium than in the absence of calcium. Spermine, a standard inhibitor of the pore almost completely reversed the effect of calcium thus indicating that the mitochondria were intact ab initio and suitable for use. Although calcium-induced pore opening was signifcantly inhibited by the VLC fraction in a concentration-dependent manner by 78.24, 88.55, 90.27 and 91.04% at 2.5, 5, 10 and 20 µg/ml, respectively, pre-incubation of mitochondria for 5, 10 and 15 minutes on ice and in the absence of calcium caused MMPT pore opening by two, fve, and four folds, respectively, at 1.25 µg/ml and two, fve and two folds, respectively at 2.5 µg/ml. Pre-incubation for these periods at 5 µg/ml caused the pore to open by two, seven and fve folds, respectively, and four, eight and four folds at 10 µg/ml, respectively. Conclusion: These fndings suggest that the bioactive agents in this fraction may fnd use in situations that require modulation of apoptosis. Key words: Alstonia boonei, apoptosis, calcium, chromatography, mitochondria, pre-incubation Correspondence: Oludele John Olanlokun, Laboratories for Biomembrane Research and Biotechnology, Department of Biochemistry, College of Medicine, University of Ibadan, Ibadan, Nigeria. E-mail: jodel72000@yahoo.com Access this article online Website: www.jbclinpharm.org Quick Response Code: This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms. For reprints contact: invoice@jbclinpharm.org Cite this article as: Olanlokun OJ, Oyebode TO, Olorunsogo OO. Effects of Vacuum Liquid Chromatography (Chloroform) Fraction of the Stem Bark of Alstonia boonei on Mitochondrial Membrane Permeability Transition Pore. J Basic Clin Pharma 2017;8:221-225. Efects of Vacuum Liquid Chromatography (Chloroform) Fraction of the Stem Bark of Alstonia boonei on Mitochondrial Membrane Permeability Transition Pore Oludele John Olanlokun, Titilope Olubukola Oyebode, Olufunso Olabode Olorunsogo Laboratories for Biomembrane Research and Biotechnology, University of Ibadan, Ibadan, Nigeria