Plant material collection
Ethnopharmacological approach was used to identify the plant under study (A. pluriseta) from Mbeere community of Embu County, Kenya. The gleaned information was further confirmed from documentation by Riley and Brokensha (1988) in The Mbeere in Kenya (ii), Botanical identity and use (18). A. pluriseta root samples were collected in an open community field, and the plant is not among the endangered plant species. Therefore, no prior permission was sought before the plant samples were collected. We collected the plant samples within GPS co-ordinates 0°46'27.0"S 37°40'54.9"E; -0.774156, 37.681908. Further authentication of plant sample identity was undertaken by Prof. S. T. Kariuki, a botanist at Egerton University, Kenya. A voucher specimen (number NSN2) was given and deposited at the same institutions herbalium.
Processing of plant samples
The plant materials were processed, extracted and finally fractionated as described in Njeru and Muema (5). Briefly, root materials were cut into small pieces and allowed to air-dry in the dark at 23 ± 2˚C until they attained a constant weight. They were thereafter ground with an electric miller (Retsch SR 200, Haan, Germany). Fifty grams of ground material was macerated in 200 mL methanol (Sigma Aldrich, St. Louis, USA) for 48 hours. This extract was filtered out using Whatmann 1 filter paper, and the process repeated once more. Both filtrates were pooled together, and methanol evaporated from the filtrate by a rotor evaporator (Laborota 4000 efficient, Heidolph, Germany). The resulting dry extract was stored at -20˚C until use. To fractionate A. pluriseta root samples, we used organic solvents of increasing polarity. Root powder (50 g) was macerated in 200 mL of petro ether solvent with intermittent shaking for 48 hours. Thereafter, the extract was filtered out. Another 200 mL petro ether (PE) was added into plant material, and the process repeated after which the two filtrates were pooled together. The resulting marc was air-dried, after which it was further extracted with solvents of increasing polarity (namely dichloromethane (DCM), ethyl acetate (EA), and finally methanol (MeOH) solvent. The organic solvent fractions were concentrated with rotor evaporator as described before (5).
In vitro cytotoxicity test
An MTT assay previously described by Njeru, Obonyo (19) was followed to evaluate the toxicity of the A. pluriseta extracts on Vero cells (from African green monkey kidney cells (Cercopithecus aethiops epithelial cell line; ATCC CCL-81)). MTT assay is a colourimetric assay pegged on the ability of mitochondrial enzyme (succinate dehydrogenase) to reduce yellow water-soluble MTT to an insoluble coloured substance (formazan) that is spectrophotometrically measurable. The amount of the formazan formed is directly proportional to the measure of cell viability. This is because only metabolically active cells can reduce MTT into formazan. The cell line grown to 70–80% confluency in a medium (containing 100 mL DMEM, 10 mL fetal bovine serum (FBS), 1 mL penicillin-streptomycin, 1 mL amphotericin B, 1 mL L-glutamine and 0.1 mL gentamycin) was incubated in the presence of sample extracts at standard conditions (37˚C in 5% CO2 ) at 1.0 × 105 cells/mL in a 96-well microtiter plate. The cells were exposed to decreasing concentrations of respective plant extracts (250 − 0.24 µg/mL for petroleum ether and dichloromethane fractions; 500 − 0.49 µg/mL for ethyl acetate and methanolic fraction). Each sample concentration was tested in duplicates for 48 hours. A post-exposure incubation of 4 hours in 10 µL of 5 mg/mL MTT solution followed the addition of 100 µL acidified isopropanol (0.04 N HCl in isopropanol). The well plates were gently shaken for 5 minutes to dissolve the formazan, and then optical density measured using ELISA Scanning Multiwell Spectrophotometer (LabSystems - Multiskan EX) at 562 nm using 690 nm as the reference wavelengths. The last column of microtiter well plate containing medium without plant extracts were included as the negative control. The percentage cell viability (%) was calculated at each concentration using the formula provided below (1, 19, 20).
Cytotoxic concentration values which represented the plant extract concentration that kills 50% of the Vero cells (CC50), was determined by regression analysis. A particular plant extract was considered cytotoxic if it had CC50 of less than 20 µg/mL and selectivity index (SI) of less than 1.0 (1, 21, 22).