Collection, identification and processing of plants
The plants, P. umbellatum (voucher number 19813) and T. benthami (voucher number 33628) were harvested in the village of Santa Mbei in the North West Region of Cameroon, based on ethnobotanical and pharmacological information. Voucher specimens were taken to the National Herbarium of Cameroon, Yaoundé, where they were authenticated by a botanist and voucher numbers assigned to them. The leaves and roots of T. benthami and the leaves, roots and seeds of P. umbellatum were air dried to constant weight and then ground to fine powder.
Preparation of crude extracts and stock solutions
Each powder was macerated for 48 h, sequentially, three times per solvent, in hexane, methylene chloride, and then methanol. The filtrates were concentrated using a rotary evaporator (BUCHI Rotavapor R-200, Switzerland) at the boiling points of the solvents. Each crude extract was recovered with a small volume of methylene chloride and left on the shelf for 72 h for any residual solvent to evaporate. The dried extracts were then weighed, and the percentage yield calculated. Stock solutions of 25 mg/mL were also prepared from each extract in dimethyl sulfoxide (DMSO, solvent grade >99.8 %, from Sigma-Aldrich, Germany) and kept at −20 °C until tested on worms and larvae.
Isolation and screens on Onchocerca ochengi adult worms
Isolation of O. ochengi adult worms (macrofilariae)
Worms were isolated by the method described by Cho-Ngwa et al. [20]. Briefly, fresh pieces of umbilical cattle skin with palpable nodules bought from slaughter houses in Douala and Buea, Cameroon, were thoroughly washed with soap and water, and the inner and outer surfaces sterilized with 70 % ethanol which was allowed to evaporate in a lamina flow sterile hood. Nodules were carefully excised, and the recovered worm masses submerged in complete culture medium (CCM, which is RPMI 1640 with NaHCO3 and supplemented with 25 mM HEPES, 0.3 g γ-irradiated L-glutamine powder, 5 % newborn calf serum, 200 units/mL penicillin, 200 μg/mL streptomycin and 0.25 μg/mL amphotericin B; pH 7.4) in 24-well culture plates (NUNC, USA). The plates containing the worms were incubated overnight at 37 °C under an atmosphere of 5 % CO2 in humidified air (in a HERACELL-150i CO2 incubator, USA). Thereafter, the viability of worms and sterility of cultures were evaluated using an inverted microscope (Nikon Eclipse TS100, China) prior to drug testing in primary and secondary screens.
Primary screens on Onchocerca ochengi adult worms
The primary screen was done in order to eliminate inactive extracts. The worms were treated in quadruplicates with either an extract at 500 μg/ml in CCM, or auranofin at10 μM (serving as positive control) [21] or 2 % DMSO (negative control), and incubated for 5 days at 37 °C in 5 % CO2 atmosphere, and their viabilities assessed. The 2 % DMSO was shown to be safe for worms in our previous studies [11–13].
Adult male worm viability was assessed through evaluation of worm motility using an inverted microscope. Activity scores ranged from 100 % (complete inhibition of motility), 75 % (only head or tail of worm shaking occasionally), 50 % (whole worm motile, but sluggishly), 25 % (only little change in motility), to 0 % (no observable change in motility).
Activity of extracts on female worms was assessed biochemically, by visual estimation of percentage inhibition of formazan (blue colour) formation following incubation of the nodules in 500 μl of 0.5 mg/ml MTT (3-(4, 5 dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, from Sigma) for 30 min [11, 12]. Activity scores assigned ranged from 100 % parasite killing (no blue formazan coloration seen), 90 %, 75 %, 50 %, 25 %, to 0 % (entire worm appears blue as in negative control).
An extract was considered active if there was ≥ 90 % inhibition of male worm motility or of formazan formation; moderately active if there was a 50–89 % inhibition of male worm motility or of formazan formation and inactive if there was a < 50 % inhibition of male worm motility or of formazan formation. All experiments were repeated once prior to the secondary screens.
Isolation of and screens with Onchocerca ochengi microfilariae
Preparation of mammalian cells
LLC-MK2 obtained from American Type Culture Collection (ATCC, Virginia, USA) were proliferated in CCM at 37 °C under an atmosphere of 5 % CO2 in humified air. The cells were grown in 96-well plates until they became fully confluent, and served as feeder layers for the microfilariae (mf) assays. The cells were also used for cytotoxicity assessment of the extracts [13].
Isolation of Onchocerca ochengi microfilariae
This was by the method of Cho-Ngwa et al. [11] with slight modifications. Briefly, umbilical cattle skin pieces containing palpable nodules were obtained from the abattoir, cleaned, carefully shaved, and sterilized with 70 % ethanol. Skin slivers were obtained and incubated for 4–6 h at room temperature in CCM and the emerged and highly motile O. ochengi mf were concentrated by centrifugation (400×g, 10 min). The mf were re-suspended in CCM and distributed into wells (about 15 mf/100 μL of CCM/well) of the 96-well plates containing the LLC-MK2 cell layer, and their viability and sterility ascertained for 24 h prior to addition of extracts.
Primary screens on O. ochengi mf
This was done as previously described [11]. Primary screens were done at 500 μg/mL in duplicates, in order to eliminate inactive extracts. The mf were incubated at 37 °C under an atmosphere of 5 % CO2 in humidified air for 5 days. Mf motility was scored microscopically, daily. The percentage motility inhibition scores were assigned as 100 % (all mf immotile), 75 % (only head or tail of mf shaking, occasionally), 50 % (whole body of mf motile but sluggishly or with difficulties), 25 % (almost vigorous motility), 0 % (vigorous motility).
Secondary screens on O. ochengi adult worms and mf
This was done as previously described [11]. Extracts with 100 % activity on both adult female worms and mf at primary screens were re-tested at serial dilutions of eight concentrations (spanning the range of 500 to 3.91 μg/mL), in order to determine the IC50 values using Graphpad prism software, version 6.0 (GraphPad Prism INC., CA, USA).
Isolation and screens on Loa loa microfilariae
Isolation of Loa loa mf
Ethical clearance (2013/11/371/L/CNERSH/SP) and administrative clearance (631–06.14) were obtained from the Cameroon National Ethical Committee and the Ministry of Public health, respectively. Local administrative clearance was given by the District Medical Officer of the Edea health district where patients were recruited for the study. Informed consent was obtained freely from individuals who tested positive for high L. loa mf load.
Isolation of L. loa mf was done as previously described by Gousard et al. [22] but with some modifications. Freshly collected L. loa -infected blood (2 mL) was diluted (1:2) with incomplete culture medium (ICM; i.e., CCM without serum) and carefully layered on 4 ml of Ficoll-pacque (TM) in a 15 mL centrifuge tube. The tube was spun in a swing bucket centrifuge (400×g, 15 min, 25 °C), and the ficoll layer containing mf recovered and washed three times with ICM. The mf were re-suspended in CCM, and then distributed in wells of a 96-well microtiter plate containing LLC-MK2 cell layers (~15 mf/well/100 μL of CCM). The mfs were monitored for 24 h for viability and sterility before treatment with extracts.
Screens on L. loa mf
Only the most active extracts on O. ochengi adult female worms and mf were screened on L. loa mf. This was done as in the primary O. ochengi mf screen and at serial dilutions of 8 concentrations (500–3.91 μg/mL).
Toxicity assessment of extracts
Cytotoxicity assessment of extracts
Cytotoxicity of extracts with good filaricidal activities were assessed on LLC-MK2 cells on day 5 as part of the mf secondary screen assays as previously described [11]. The 50 % cytotoxic concentrations (CC50 values) for these cells were determined by microscopic examination. Dead or deformed cells were usually detached from the bottom of the vessel and were rounded in shape. The selectivity index (SI) of each extract was calculated as the ratio of CC50 of the extract on the mammalian cells to the IC50 of the extract on the O. ochengi or L. loa parasite.
Acute toxicity
The acute toxicity was evaluated for the most active extracts on O. ochengi adult worms and mf in accordance with the Organisation for Economic Co-operation and Development (OECD) guidelines for testing chemicals [23]. Ethical permit was obtained from the University of Buea, Faculty of Science Institutional Animal Care and Use Committee (IACUC). Eight nulliparous and non-pregnant Balb/c mice, ten weeks old (averagely 18 g each) were divided in four groups (2 per group) and kept in their cages for 5 days to allow for acclimatization to the new housing conditions. Three groups were used for the three most active extracts and the last group as negative control. Food, but not water was withheld for 4 h after which the animals were weighed and the extract administered orally using a gavage needle at a limit dose of 2000 mg/kg body weight with a volume of 1 mL/100 g body weight of mice. The extracts were diluted with distilled water prior to administration, while negative control mice received the vehicle (2 % DMSO diluted in distilled water, 200 μL/kg body weight). After administration of the extracts, food was withheld further for 2 h. The animals were observed individually after dosing, once every 30 min during the first 4 h. The animals were weighed every 2 days and observed for physical activity and behavioural pattern, food and water intake, changes in skin and fur, eyes and mucous membranes, tremours, convulsions, salivation, lethargy, sleep, coma and death for 14 days.
Phytochemical screening
Phytochemical derivatives in the 3 most active extracts were investigated by standard methods against known references. The presence of saponins and sterols was determined as described earlier [24]. Saponins were detected by dissolving trace amounts of the extract in distilled water and shaking thoroughly. Frothing which persisted on warming was observed and taken as evidence. The presence of steroids was determined by dissolving 0.1 g extract in methylene chloride (3 mL) and acetic anhydride (2 drops). The mixture was boiled and 1 drop of concentrated sulphuric acid added after cooling. A green colour indicated the presence of sterols. For tannins, 1 g of extract was dissolved in methanol and 2 drops of ferric chloride were added, and the mixture observed for appearance of an olive green colour (which is indicative of the presence of tannins) [8]. Alkaloids were tested by thin-layer chromatography (TLC) on aluminium plates coated with silica gel. Each extract (0.5 g) was dissolved in methylene chloride, spotted on the TLC plate, and resolved using a mobile phase constituted of 20 % hexane/ethylacetate. Resulting spots were developed by spraying the plate with Drangedoff’s reagent, and shiny yellow spots indicated the presence of alkaloids [25]. The presence of flavonoids was revealed by formation of crimson red colour when 0.1 g extract is dissolved in methanol with few fragments of magnesium ribbons and 1 drop of concentrated hydrochloric acid [25].
Statistical analysis
To determine IC50 values of active extracts, the filaricidal activity data obtained were analysed using GraphPad Prism 6.0 (GraphPad Prism INC., CA, USA). The logarithm of the extract concentration was plotted against its activity determined by microscopy, to obtain a nonlinear regression curve –fitting and a variable slope sigmoidal dose- response curve.