Plant material and extraction
F. heitzii ( Rutaceae), G. lucida (Guttiferae) and H.lyrata (Euphorbiaceae)were harvested in the East region, Ebolowa andBatchingou in Cameroon. The plants were identified at the Cameroon NationalHerbarium where voucher specimens were deposited under the reference number1441/HNC, 53354/HNC and 32301/HNC respectively for F. heitzi, G. lucida andH. lyrata.
Each collected sample (leaves and barks for G. lucida, roots and barksof H. lyrata, and fruits and roots for F. heitzii) was driedat room temperature (28 ± 3°C), pulverized and powdered. Each powder(50 g) was macerated in 500 ml of methanol or water for 72 h atroom temperature. After 72 h, the mixture was filtered using a paper filterWhatman No. 1. Each filtrate was then concentrated under vacuum (Rotaryevaporator, Heidolph WB 200) to obtain the crude extract. Each crude extractobtained was then weighed and stored at 4°C.
Cytotoxic activity assay
Cell lines and treatment
The effect of the extracts and compounds on cell growth was determined in apanel of human tumor cells including lung A549 adenocarcinoma, breastcarcinoma MCF-7, prostate carcinoma PC-3, cervical carcinoma HeLa and acutemonocytic leukemia cell line THP-1, obtained from National Cancer Institute,USA. THP-1, A-549 and PC-3 were maintained in RPMI medium while MCF-7 andHeLa were cultured in MEM medium. All media used were supplemented with 10%fetal bovine serum (FBS), 100 IU/ml penicillin. The cell lines weremaintained under standard cell culture conditions at 37°C and 5%CO2 in a humidified environment.
Cytotoxic activity by SRB assay
In vitro cytotoxicity against above mentioned five human cancercell lines was determined using sulphorhodamine B assay (SRB) as describedpreviously . Briefly, cells were harvested in log phase using trypsin (0.05%trypsin, 0.02% EDTA, in PBS). The cell suspensions were diluted withappropriate growth medium to obtain the cell densities depending on the cellline: (104 cells/well for HeLa, 104 cells/well forA549, 104 cells/well for THP-1, 1, 5×104cells/well for MCF-7 and 104 cells/well for PC-3). An aliquot of100 μl of each suspension were seeded in 96 wells cell cultureplates. The cells were incubated at 37°C in an atmosphere of 5%CO2 and 95% relative humidity in a CO2 incubator.After 24 h incubation, test materials (100 μl/well) atvarying concentrations (1, 10, 30 and 50 or 100 μg/ml) were addedto the wells containing cells. Paclitaxel 0.1, 1 and 10 μM wasused as positive control. Suitable controls with equivalent concentration ofDMSO were also included. The plates were further incubated for 48 h ina CO2 incubator after addition of test material. After incubationcells were fixed by gently layering trichloroacetic acid(50 μl/well, 50% w/v) on top of the medium in all the wells andincubated at 4°C for 1 h. The plates were washed five times withdistilled water and air-dried. Cell growth was measured by staining withsulforhodamine B dye (0.4% w/v in 1% acetic acid,100 μl/well). The unbound dye was washed 3–5 times with 1%acetic acid and plates were air dried. The adsorbed dye was dissolved inTris-Buffer (100 μl/well, 0.01 M, pH 10.4) and plateswere gently shaken for 10 min on a mechanical shaker. The opticaldensity (OD) was recorded using a 96 well plate reader. Growth inhibitionwas calculated by subtracting mean OD values of respective blank from themean OD value of experimental set. Percentage growth in presence of testmaterial was calculated considering the growth in absence of any testmaterial as 100% and in turn percentage growth inhibition in presence oftest material was calculated. The viability and growth in the presence oftest material is calculated by following formula.
IC50 value is the concentration of sample required to inhibit 50%of the cell proliferation and was calculated by plotting the percentagesurvival versus the concentrations, using Microsoft Excel. For all samples,each compound concentration was tested in triplicates in a singleexperiment.
Microbial growth conditions
A total of ten microbial strains were tested obtained from the American TypeCulture Collection for their susceptibility to extracts and compounds. Thesestrains comprised of three yeasts: Candida albicans (ATCC 90028),Candida krusei (ATCC 6258), and Candida tropicalis(ATCC 750); one filamentous fungi: Aspergillus fumigatus (MTCC1811); three Gram-negative bacteria: Pseudomonas aeruginosa ATCC27853, Escherichia coli ATCC25292, vancomycin-resistantEnterococcus faecalis (VRE) and three Gram-positive bacteria:Staphylococcus aureus ATCC 29213, methicillin-resistantStaphylococcus aureus (MRSA, ATCC 33591) and Staphylococcusepidermidis (ATCC 12228). They were maintained on agar slant at4°C and sub-cultured on a fresh appropriate agar plates 24 h priorto any antimicrobial test. The Mueller Hinton Agar (MHA) and Sabourauddextrose Agar (SDA) were used for the activation of bacteria and fungirespectively. The Mueller Hinton Broth (MHB) and RPMI 1640 were used for theMIC determinations.
Suspensions of bacteria and yeasts were prepared in sterile normal saline(0.85%) from 24 h grown on SDA or MHA at 37°C. The turbidity ofthe microbial suspension was adjusted with a densitometer to a McFarlandstandard of 0.5 for bacteria and 0.9 for yeast, which are equivalent to1–5 × 108 CFU/ml and 1–5 ×107 CFU/ml respectively.
Inoculum suspensions of Aspergillus species were prepared fromfresh, mature (3 to 5 days old) cultures grown on Sabouraud agar orpotato dextrose agar slants. The colonies were covered with approximately5 ml of distilled containing 5% Tween 20. Then, the suspensions weremade by gently probing the colony with the tip of a Pasteur pipette andtransferred to a sterile tube; the resulting suspensions were homogenizedfor 15 s with a vortex mixer at 2000 rpm. The suspension wasfiltered and collected in a sterile tube. The inoculum size was adjusted to1-5 × 106 spores/ml by microscopic enumeration with acell-counting hematocytometer. Adjusted suspensions were checked by plating0.01 ml of a 1:100 dilution onto PDA plates to determine the viablenumber of CFU/ml. The plates were incubated at 37°C and observed dailyfor the presence of fungal colonies. The colonies were counted as soon aspossible after the observation of visible growth.
The MIC was performed by broth microdilution method, with Mueller HintonBroth (MHB) for bacteria and RPMI 1640 medium (containing L-glutamine,without sodium bicarbonate and buffered to pH 7.0 with 0.165 Mmorpholine propanesulfonic acid) for fungi. Stock solutions of extracts wereprepared in 100% dimethylsulfoxide (DMSO; Sigma) and twofold serialdilutions were prepared in media in amounts of 100 μl per well in96-well. The above-mentioned microbial suspensions were further diluted to1:100 in media, and a 100 μl volume of this diluted inoculum wasadded to each well of the plate, resulting in a final inoculum of1.5×106 cfu/ml for bacteria,1.5×104 cfu/ml for A. fumigatus and1.5×105 cfu/ml for yeasts. The final concentrationof samples ranged from 7.8-1000 μg/ml. The medium without theagents was used as a growth control and the blank control used containedonly the medium. Ciprofloxacin and Amphotericin B served as the standarddrug controls. The microtiter plates were incubated at 37°C for24 h, 48 h and 72 h respectively for bacteria, yeasts andAspergillus species. The plates were read visually, and the MICwas defined as the lowest concentration of the antifungal agents thatprevented visible growth with respect to the growth control.
The one-way ANOVA at 95% confidence level was used for statistical analysis.