Collection of Plant Stem bark, Leaf and Fruit
The plant parts used in this study were selected based on ethnopharmacological
information as antimalarial plant used in southern part of Nigeria. Allanblackia floribunda leaf, stem bark and seed were collected during the period of January and July, 2016, from a forest area at Ohogua community, Ovia East Local Government, Benin City, Edo Sate, Nigeria. The plant was authenticated by a Botanist at the Department of Plant Biology and Biotechnology, Faculty of Life Sciences, University of Benin, Nigeria and voucher specimen of the sample (UBHA361) was deposited at the herbarium of the same department.
Preparation Of Extract
100 g of the macerated A. floribunda stem bark and leaf were soaked in 1000 mL of absolute methanol for 72 h with occasional stirring. The extracts were then filtered using double layered muslin cloth and the filtrate concentrated to dryness by using a rotary evaporator at reduced pressure while the oil was obtained from about 200 g of the powdered seed using hot water floatation method. The extracts obtained were stored at 4⁰C until used.
Fractionation of A. floribunda stem bark extract
Fractionation of the stem bark extract was done using solvent of increasing polarity as described by Hassan et al. (2012). The methanol stem bark extract of A. floribunda was partitioned into fractions of n-hexane, dichloromethane, ethyl acetate and hydromethanol. The fractions obtained were concentrated using a rotary evaporator (RE 300, Bibby Scientific, UK) with reduced pressure at 45˚C and final concentrate was obtained using silica gel.
Qualitative Phytochemical Screening and antioxidant activity of A. floribunda stem bark Fractions
The qualitative test for phytochemicals present in fractions were carried out using standard procedures [7, 8]. The free radical scavenging capacity of the fractions against 1,1–diphenyl–2–picrylhydrazyl (DPPH) radical was determined by a modified method of Brand-Williams et al. . Phosphomolybdate reduction capacity was estimated using the method described by Prieto et al. .
Cultivation Of Parasites
Trager and Jensen procedure was used to culture P. falciparum [11, 12]. The Plasmodium strain (Pf3D7) used was chloroquine sensitive isolated from Nigeria and India, respectively. The parasites were cultured in O + RBC as host cells and maintain in RPMI 1640 medium supplemented with gentamicin solution 0.01 mg/mL, 25 mM HEPES buffer, 25 mM NaHCO3 and 1% Albumax II maintained in 5% CO2 and incubated at 37 °C. Parasitaemia was determined using light microscopy (Giemsa stain).
In Vitro Antiplasmodial Activity
The growth inhibition of chloroquine-sensitive Plasmodium falciparum strain (3D7) by the plant extracts was evaluated by means of the Mark III test, as developed by the WHO . Filter sterilized extracts (25, 12.5, 6.25, 3.12,1.56, 0.78 and 0.39 µg/ mL) were incorporated in 96-well plate containing 200 µL of 0.5% P. falciparum culture that had been synchronized and diluted to 3% hematocrit with red blood cells. Wells with parasitized red blood cells without plant extract serve as negative controls whereas wells containing cultures with chloroquine diphosphate serve as positive control. Parasitaemia was evaluated after 48 h by Giemsa stain and the average suppression of parasitaemia was calculated.
Average % suppression =
Average % parasitaemia in control ₋ Average % parasitaemia in test
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Average % parasitaemia in control
The antiplasmodial activities of the extracts were expressed using IC50. The IC50 is the inhibitory concentrations of the drug/extracts that induced 50% reduction in parasitaemia compared to control (100% parasitaemia). It was determined using SPSS statistical tool pack based on interpolation from the parasite growth inhibition curve generated from each plant extract. Each sample was tested in duplicate and the IC50 obtained were pooled and expressed as geometric means and standard deviations. The independent sample t-test was used to compare mean IC50 of antimalarial activity between plant extracts using STATA version 13 (Software Stata Corp, College Station, TX, USA). Finally, the in vitro antiplasmodial activity was rated in accordance with the system of antiplasmodial activity of Rasoanaivo et al. .
Cytotoxicity Test (cell Viability Assay)
Monkey kidney epithelial cell (LLC MK2) were used to assess the cytotoxicity of the most active plant extract. Vero cells were maintained in Dulbecco’s Modifed Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum FBS, glutamine (2 mM), penicillin (100 units / mL) and streptomycin (100 µg / mL). The cells were seeded in 96 well plates at 10,000 cells per well in 100 µL culture medium. The cells were incubated at 37⁰C in a humidified 5% CO2 atmosphere. After 48 h, the medium was discarded by inversion of the microtitre plate and thereafter replaced with 100 µL of fresh culture medium followed by 100 µL of crude plant extract at a concentration of 1000 µg / mL in row H of the 96 well plate and serially diluted twofold to give concentrations ranging from 500 to 7.81 µg / mL. Row A. of the 96 well plate serve as control wells, whereby the negative control contained culture medium and the LLC-MK2 cells without plant extract (Maximal cell growth). The cells were maintained at 37 in a CO2 incubator before determining their viability by using MTT assay as described by Mosmann . The absorbance for each well was measured between 490–630 nm in a micro-titre plate reader and the percentage cell viability (CV) was calculated using the formula:
CV = Average Absorbance of dublicate drug wells x 100
Average absorbance of control well
A dose-response curve was plotted to calculate the concentration that destroyed 50% of the Vero cells (CC50).
Gas Chromatography (gc) Analysis Of Most Active Fraction (n-hexane)
The flavonoids, terpenes, alkaloids, terpenoids, quinones and volatile organic constituents profile of the n-hexane fraction was determined using gas chromatography with flame ionization detector. Gas chromatography analysis was carried out on a HP 6890 Powered with HP ChemStation Rev. A 09 01 Software.
The statistical analyses of the results were carried out. The various results obtained from this study were expressed as mean ± SEM. One way analysis of variance (ANOVA) followed by Tukey's HSD (honest significant difference) test was used to determine significance differences between the groups. Statistical significance was declared when P value was less than 0.05. The statistical analysis was performed using the statistical package for social science (SPSS) for windows, version 16.0.