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dc.contributor.authorIWAKUN, T. I.-
dc.date.accessioned2019-03-25T15:26:56Z-
dc.date.available2019-03-25T15:26:56Z-
dc.date.issued2012-06-
dc.identifier.urihttp://adhlui.com.ui.edu.ng/jspui/handle/123456789/850-
dc.descriptionA Thesis in the Department of Biochemistry submitted to the Faculty of Basic Medical Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy of the University of Ibadan, Nigeria.en_US
dc.description.abstractInhibition of Ca²⁺ sarco/Endoplasmic Reticulum Ca²⁺-ATPase (SERCA) could result in prolonged elevation of cytosolic Ca²⁺ concentration, opening of Mitochondrial Membrane Permeability Transition (MMPT) pore, release of cytochrome C and ultimately apoptosis. This phenomenon provides a rational approach for drug development. Kolaviron, a biflavonoid complex from Garcinia kola (Heckel) seeds has been shown to elicit some pharmacological activities. The effects of kolaviron on MMPT and its four biflavonoids on SERCA la (an isoform of SERCA) were investigated. Garcinia kola seeds were dried, powdered and subjected to soxhlet extraction to obtain kolaviron and its biflavonoids were separated using HPLC. Their molecular weights were determined by mass spectroscopy, while the structure of a newly identified biflavonoid was determined by Nuclear Magnetic Resonance (NMR). Rabbit hinge muscle SERCA Ia and rat liver mitochondria were prepared by differential centrifugation and the extent of opening of MMPT pore was measured using spectrophotometer at 520 nm. ATPase activity was measured by coupled-enzyme assay technique. Structural conformational changes of SERCA in the presence of biflavonoids were investigated using time-resolved fourier transfer infrared technique. Data were analysed using ANOVA at P= 0.05. Kolaviron had garcinia biflavanones (GB); GBI, GB2, kolafIavanone and a newly identified biflavonoid GB-2H-glycoside. These biflavonoids (40uM) inhibited SERCA la with IC₅₀ values of 17 uM (GB2), 42 uM (GBI), 49 uM (kolaflavanone), and >80 uM (GB-2H-glycoside). The biflavonoids reduced maximum activity (Vmax) of SERCA by 55.9% (GBI), 65.6% (GB2), 47.2 % (kolaflavanone) and 32.5 % (GB-2H-glycoside), but not the affinity (Km). Kinetic analysis suggests that these biflavonoids exhibited a non-competitive mode of inhibition. Investigation of ATP hydrolysis in the presence and absence of GB2 (most potent inhibitor) showed spectra with a negative band at 1254 cm⁻¹ and a positive band at 1091 cm⁻¹, which are characteristics of ATP hydrolysis by SERCA. Studies on conformational changes of SERCA in the presence of absence of 10mM GB2 showed negative bands at 1662 and 1407cm⁻¹, and positive bands at 1632 and 1487 cm⁻¹, indicating ATP binding to the enzyme. Also the presence of positive band at 1718 cm⁻¹ showed formation of aspartyl phosphate, thus indicating that GB2 does not prevent ATP binding and protein phosphorylation. Studies of Ca²⁺ release by SERCA into the sarcoplasmic reticulum lumen in the presence of 2 mM GB2 and 10 mM caged-ATP showed the absence of a negative band at 1572 cm⁻¹; this band is the marker band for Ca²⁺ release from carboxylate group. Oral administration of varying doses of kolaviron to rats induced the opening of MMPT pore in a dose-dependent manner in the absence of Ca²⁺ by 2.0, 2.5 and 6.6 folds at 100, 150 and 200 mg/kg, respectively. Kolaviron synergistically induced the opening of MMPT pore in the presence of CA²⁺ by 22, 25 and 24 folds at 100, 15O and 200 mg/kg, respectively. Opening of mitochondrial membrane permeability transition pore indicates that the biflavonoid from kolaviron has potential to target sarco\endoplasmic reticulum Ca²⁺-ATPase for drug development in diseases associated with dysregulated apoptosis.en_US
dc.language.isoenen_US
dc.subjectKolavironen_US
dc.subjectGarcinia biflavanone 2en_US
dc.subjectCellular Ca²⁺ transporten_US
dc.subjectSarco/endoplasmic reticulum Ca²⁺ -ATPaseen_US
dc.titleMODULATION OF CELLULAR CALCIUM TRANSPORT BY BIFLAVONOIDS PURIFIED FROM SEEDS OF GARCINIA KOLA (HECKEL)en_US
dc.typeThesisen_US
Appears in Collections:Theses in Biochemistry

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