Chemicals
Sodium dodecyl sulphate, bovine serum albumin (BSA), sodium chloride (NaCl), MgCl2·6H2O, acetylthiocholine iodide (ATCI), galantamine, 5,5-dithiobis-2-nitrobenzoic acid (DTNB), acetylcholinesterase (AChE) enzyme from electric eels (type VI-S lyophilized powder), sodium nitrite, ferrous sulfate, indomethacin and 15-lipoxygenase from Glycine max purchased from Sigma (Germany) and Tris(hydroxymethyl)aminomethane from Sigma, (Switzerland). Foetal calf serum (FCS), penicillin/streptomycin/fungizone (PSF) and Dulbecco’s modified Eagle’s medium (DMEM) were obtained from Highveld Biological Products (South Africa). Phosphate buffered saline (PBS) and trypsin were purchased from Whitehead Scientific (South Africa). Quercetin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) were purchased from Sigma-Aldrich St. Louis, MO, USA.
Limonoids compounds
The three limonoids compounds (dregeanin DM4, rohituka 3 and trichilia lactone D5) studied in this work were isolated from seeds of Trichilia welwitschii. We previously described the isolation procedure and the structure elucidation of the compounds [9]. Chemical structures are shown in Fig. 2.
Cytotoxicity
Cell culture
Cells lines including human monocytic THP-1, murine macrophage RAW 264.7 and the Vero monkey kidney cell lines were obtained from the American Type Culture Collection (Rockville, MD, USA). They were maintained in DMEM supplemented with 10 % fetal calf serum (FCS) and 1 % penicillin/streptomycin/fongizone (PSF) under standard cell culture conditions at 37 °C and 5 % CO2 in a humidified environment.
MTT assay
The cytotoxicity of compounds was determined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium (MTT) assay as previously described [23]. The selectivity index (SI) values were calculated by dividing cytotoxicity LC50 values of normal Vero cells by the LC50 of cancer cells in the same units.
Nitric oxide inhibitory activity and viability of LPS-activated RAW 264.7 macrophages
The RAW 264.7 macrophages cells were seeded in 96 well-microtitre plates and were activated by incubation in medium containing 1 µg/mL LPS alone (control) or lipopolysaccharide with different concentrations of the samples dissolved in DMSO. Quercetin served as a positive control NO inhibitor for the reduction of NO production [18].
Measurement of nitrite
Nitric oxide released from macrophages was determined by measuring the nitrite concentration in culture supernatant using the Griess reagent. After 24 h incubation, 100 µL of supernatant from each well of cell culture plates was transferred into 96-well microtitre plates and an equal volume of Griess reagent was added. The absorbance of the resultant solutions was determined on a BioTek Synergy microplate reader after 10 min at 550 nm. The concentrations of nitrite were derived from regression analysis using serial dilutions of sodium nitrite as a standard. Percentage inhibition was calculated based on the ability of compounds to inhibit nitric oxide formation by cells compared with the control (cells in media without compounds), which was considered as 0 % inhibition.
Cell viability
To determine whether the observed nitric oxide inhibition was not due to cytotoxic effects, MTT assay was also performed on the macrophage cells as previously described [23].
Acetylcholinesterase inhibition activity
Inhibition of acetylcholinesterase activity was determined using Ellman’s colorimetric method [24] with slight modifications. In a 96-well plate was placed: 25 µL of 15 mmol/L ATCI in water, 125 µL of 3 mmol/L DTNB in Buffer A (50 mmol/L Tris–HCl, pH 8.0, containing 0.1 mol/L NaCl and 0.02 mol/L MgCl2·6H2O), 50 µL of Buffer B (50 mmol/L, pH 8, containing 0.1 % bovine serum albumin) and 25 µL of samples (serially diluted: 500–3.9 µg/mL for extract and fraction and 100–0.78 µg/mL for compounds and galantamine used as AChE standard inhibitor). Then, AChE (0.2 U/mL) was added to the wells and the absorbance was determined spectrophotometrically (BioTek Synergy microplate reader) at 405 nm. Distilled water was used as negative control. The percentage of inhibition was calculated as follow:
$$\begin{aligned}{\text{V}} &= \Delta_{\text{Abs}} /\Delta_{\text{t}} , \\ \% {\text{enzyme activity}} &= \left( {{\text{V}}/{\text{V}}_{ \text{max} } } \right) \times 100, \\ \% {\text{enzyme inhibition}} &= 100 - \% {\text{enzyme activity}}\end{aligned}$$
where V is the rate of the reaction in the presence of inhibitor and Vmax is the reaction’s rate of the control without inhibitor. The IC50 values of samples leading to 50 % inhibition were calculated by plotting the percentage of inhibition against the concentrations.