Collection and identification of plant materials
Various plant parts (leaves, barks and roots) of C. laurinum were collected from Finge village of the Bambui Health District in the North West Region of Cameroon in June 2010, while M. lucida plant parts were collected from Buea at the foot of Mount Fako, Cameroon in January 2011. The plants were selected based on ethnopharmacological information about them. The plants were identified and authenticated by Mr. Paul Mezili of the National Herbarium, Yaounde, Cameroon and given the voucher number (Poir) Benth No 3781/SRFK for C. laurinum and Benth No 2615/SRFK for M. lucida. Locally, C. laurinum is called “sarkaatari”.
Preparation of crude extracts and chromatographic fractions
All the plant parts collected were air dried, then ground to fine powder. The ground materials were weighed and sequentially submerged and macerated for a total of 72 hours in three solvents thus: hexane (HEX), methylene chloride (MC), and methanol (MeOH). For each solvent, the maceration was repeated twice. The mixture was filtered and the filtrate concentrated using a rotary evaporator (BUCHI Rotavapor R-200, Switzerland) at appropriate temperatures. The concentrate were recovered with methylene chloride and allowed to stand open at room temperature until all the residual solvents had evaporated. The dried crude extracts were stored at -20°C until needed for the assays.
Bioassay-guided fractionation was done on the most active crude extracts. Each of the latter extract was fixed on celite and fractionated using vacuum liquid chromatography on silica gel and then eluted with a continuous gradient of ethyl acetate (EtOAc [0–80%]) in hexane, followed with a gradient of methanol (MeOH [0-40%]) in methylene chloride. Collected fractions were pooled on the basis of their thin layer chromatographic (TLC) profiles.
Isolation and culture of O. ochengiadult worms (macrofilariae)
This was done by the method of Cho-Ngwa et al.  with slight modifications. Briefly, fresh pieces of umbilical cattle skin containing large amount of palpable nodules were brought from local slaughterhouses to the laboratory. The skin was thoroughly washed successively with tap and distilled water, drained and totally covered with 70% ethanol which was allowed to evaporate on its own in a laminar flow hood sterile environment. Pale orange-yellow O. ochengi adult worm masses containing essentially viable adult female and male worms were recovered by careful dissection of the nodules using sterile razor blade. The extracted worms were immediately submerged into complete culture medium (CCM) (RPMI-1640 supplemented with 25 mM HEPES, 2 g/L sodium bicarbonate, 20 mM L-glutamine, 10% new born calf serum [SIGMA, USA], 200 units/ml penicillin, 200 μg/ml streptomycin and 2.5 μg/ml amphotericin B [Sigma, USA], pH 7.4) in 24 wells. The viability of worms was determined by microscopic examination using an inverted microscope (euromex, Holland). The cultures were incubated at 37°C under an atmosphere of 5% CO2 in humidified air in a HERACELL-150 CO2 incubator (Thermo Electron, Germany) for 05 days post addition of drug.
Extraction of microfilariae
Fresh and cleaned infected cattle skin was sterilized as above and tautly attached onto a sterile wooden board. A portion of the skin was then carefully shaved with razor blade, rinsed with distilled water and completely covered with 70% ethanol. The ethanol was allowed to evaporate freely and completely in a laminar flow hood. The skin was then firmly attached onto an autoclaved cylindrical piece of wood and close crisscross cuts of about 0.5-1 mm apart were made into the epidermal and dermal layers, over the entire skin surface. The entire assembly was transferred into a sterilized glass cylinder and an appropriate volume of CCM was added to just cover the entire skin. The assembly was incubated for 4 – 6 hours at room temperature. The emergent and highly motile microfilariae (mfs) were concentrated by centrifugation (400 × g for 10 minutes) and quantified microscopically. Then, 100 μl of CCM containing 15–20 mfs was transferred into each well of a 96 well microtitre culture plates. After addition of drugs, the mfs were cultured at 37°C under an atmosphere of 5% CO2 in humidified air in a HERACELL-150 CO2 incubator (Thermo Electron, Germany).
Preparation of mammalian cells for microfilarial cultures and cytotoxicity assay
Monkey kidney epithelial cells (LLCMK2) purchased from the American Type Culture Collection (ATCC, Virginia, USA) was proliferated in complete culture media at 37°C and 5% CO2. Once the cells became fully confluent, the old media was decanted and the cells were dislodged with 0.125% trypsin and 0.5 mM EDTA in serum-free RPMI-1640 culture medium. The dislodged cells were re-suspended in 10 mL of complete culture medium and centrifuged at 560 × g for 10 minutes to get rid of the trypsin. The last procedure was repeated once. The cell suspension (100 μL/well) was transferred into 96-well microtitre culture plates and kept in the CO2 incubator for cells to grow and become fully confluent, usually taking 3–5 days depending on the initial concentration of cells. LLCMK2 cells served as feeder layer for mfs cultures and also for cytotoxicity studies. The selectivity index (SI) of each extract was calculated as the ratio of CC50 of drug to these mammalian cells to the IC50 of the drug on the parasites.
Preparation and in vitro screening of crude extracts and fractions
A stock solution of 25 mg/ml crude extract in ≥99.9% sterile dimethyl sulfoxide (SIGMA, USA) was prepared. Adult worm assays were conducted in 24-well plates (2 mL/well) (NUNC, USA) for 5 days without any change of medium. On the other hand, all mfs-only assays were conducted in 96-well microtitre plates (200 μL /well) that contained confluent feeder layer of the monkey kidney cells. The media used in preparing the feeder cell layer was removed by swift decantation before fresh medium and worms for drug testing were added. Assays were run for 5 days post-addition of extract without any change of medium.
Primary screen for adult worm and microfilariae
This was done in order to eliminate inactive extracts. Each extract was dissolved to 2X its final test concentration in CCM in a single sterile well before distribution into replicate wells. In this regard, 1 ml per well or 100 μl per well for adult worm or mfs respectively, were transferred into replicate culture wells, each already containing either adult worm in 1 ml of CCM or mfs in 100 μl of CCM, to give a final volume of either 2 ml for the adult worm assay or 200 μl for the mfs assay. The crude extracts were tested in quadruplicate wells (for adult worms) and in duplicate (for mfs) at a single concentration of 500 μg/mL. A compound (BTU55261) known to be active (unpublished) was used as the positive control for the adult worm assay, while ivermectin (IVM) was used as a positive control for the mfs assay. BTU55261 produces 100% inhibition of formazan formation in adult worms on day 5 at a concentration of 2.5 μg/ml, while IVM totally inhibits mfs motility at 10 μg/ml by day 5. Negative controls were treated in the same way as the test samples, received the drug diluents (≤2% DMSO in RPMI), but with no drug added.
Male worm motility inhibition was scored using an inverted microscope and was recorded as 100% (complete inhibition), 75% (only head or tail of worm moving or vibrating), 50% (worm sluggish) or 25% (little change in motility), 0% (no observable inhibition of motility). Biochemical evaluation of adult worm viability was done using the MTT/formazan colorimetric assay [30, 31]. The worms were placed under sterile conditions in a well of a 48-well plate containing 500 μL/well of 0.5 mg/mL MTT (Sigma, USA) in serum free media and then incubated under the culture conditions for 30 minutes. Thereafter, the worms were blotted individually on tissue paper and colour development of the test compared with the controls. MTT, a pale yellow compound is reduced to a dark blue product, formazan by the living cells of the worms. Adult worm viability was taken as mean percent inhibition of formazan formation relative to negative control at 120 hours post addition of drug. Inhibition of formazan formation by extracts relative to the negative controls showed that an extract was either active (90% or greater mean inhibition of formazan formation), moderately active (50 - 89% mean inhibition of formazan formation) or inactive (<50% mean inhibition of formazan formation). Viability of mfs was assessed by reading their mean motility scores every 24 hours (post addition of drug) for 120 hours on the same scale as for adult male worms. An extract was considered active on the mf if there was a 100% reduction in mfs motility; moderately active if there was a 50-99% mean reduction in mfs motility and inactive if there was less than 50% mean inhibition of motility compared to untreated controls of the same experiment.
This was done in order to confirm the activity of extracts that were promising in the primary screen, as well as to determine the IC50 and selectivity index (SI) values. All the active extracts that showed 100% macrofilaricidal and/or microfilaricidal properties were re-tested as in the primary screens and at serial dilutions of 8 different concentrations starting from 500 μg/mL down to 3.91 μg/mL. The graphical analysis was done using Microsoft Excel 2007 (Microsoft Corporation, USA).
Acute toxicity of active extracts in mice
The test was conducted following the European Community Guidelines for protection of animals used for experimental purposes  and respecting the 3Rs (Replacement, Refinement and Reduction) of Animals in Research . Currently, there is no law governing the use of Balb/C mice for research purposes in Cameroon. Mice used in the study were commercially gotten from Centre Pasteur, Yaoundé. Nulliparous, non-pregnant 10 weeks old female Balb/c mice were weighing 22.4 ± 1.5 g were randomly selected and kept in their cages for 5 days before dosing . The test was done for the methanolic extracts of C. laurinum and M. lucida as well as for fractions that demonstrated an IC50 of ≤ 31.25 μg/ml on mfs. The equivalent of 5 times IC50 of each substance was injected intraperitoneally into separate groups of 6 mice while 6 control mice were injected with an equal volume of diluent (2% DMSO in normal saline). Briefly, considering the 5 × IC50s of 93.75 μg/ml for the C. laurinum extract, 10.3 mg of the extract was dissolved in 100% DMSO and then diluted in normal saline such that the final DMSO concentration was 2%. The diluted extract was then injected into the chosen mice. The same procedure was repeated for all the selected active fractions. Following dosing, the animals were observed every 30 minutes for the first 4 hours. Thereafter, the physical activity, weight of food and the volume of water consumed, changes in skin and fur, tremors, convulsions, diarrhea, sleep, coma and death were noted daily for the 14 day study period.
Following screening, phytochemical derivatives found in the most active fractions were investigated by standard methods . Briefly, the presence of alkaloid was determined using the Drangedoff’s reagent while the presence of sterols and triterpenes was determined by the Liebermann Buchard reaction. For the presence of flavonoids, the extract was dissolved in methanol and 2–3 mL of concentrated HCl was added. A spatula full of magnesium turnings was then added and the mixture observed for effervescence. The presence of saponins was determined by dissolving a trace amount of the extract in water and shaking thoroughly. Frothing which persisted on warming was observed and taken as preliminary evidence. Test for Tanins was done by adding 10 ml of distilled water to 2 g of plant extract, stirred, filtered and ferric chloride later added to the filtrate.