Collection of plant material
H. zeyheri tubers were collected in Hwange District, northwest Zimbabwe, in July 2016 after seeking permission from the Hwange Rural District Council and local community leadership. Plant identification was done by Anthony Mapaura, Senior Research Officer at the National Herbarium and Botanic Gardens of Zimbabwe, and a voucher specimen is deposited there (Voucher: CASSRGH108539).
Preparation of the sample included washing, slicing and air drying of the tubers at room temperature (25°C) over a period of 5-8 days. Extraction was done guided by Do et al. (2014), with minor modifications. The dried tubers were ground into powder and 10 g of powder were placed in 100 ml of each of 3 different solvents (ethyl acetate, ethanol and water). The mixtures were left for 24 hours (h) at room temperature. The extracts were then filtered, and the filtrates were left standing at room temperature for 24 to 48 h to dry to a sticky or powdery substance (Handa et al. 2008). Water extracts were evaporated at 55°C in a ventilated oven for up to 4 days. All the crude extracts were then stored at 4°C in a cold room until use.
The extracts of H. zeyheri were subjected to screening for antioxidant activity by two methods namely DPPH and ABTS free radical scavenging.
DPPH radical scavenging assay
The determination of the DPPH free radical scavenging activity of the crude extracts was done using the Shimada et al. (1992) method, with a few modifications. The crude extracts (40µL) were serially diluted in methanol in a 96-well plate followed by addition of a DPPH solution (160µL) freshly prepared at 25µg/ml in methanol. The plates were incubated at room temperature (25°C) in the dark for 30 minutes and the absorbance was measured on a microplate reader (Epoch, Biotek) at the wavelength of 517nm (Shimanda et al. 1992). The radical scavenging activity was calculated by the following equation:
DPPH radical scavenging (%) = equation 1.
where A0 is the absorbance of the control and A1 is the absorbance of the sample.
Ascorbic acid was used as the positive control while the mixture of methanol with DPPH solution was used as the negative control. The blanks were made of methanol and the extracts. The IC50 values were calculated as the concentration of sample required to scavenge 50% of DPPH free radicals.
ABTS radical scavenging assay
The determination of ABTS radical scavenging activity was done using the ABTS cation decolourisation assay described by Re et al. (1999), with a few modifications. The ABTS radical cation (ABTS•+) was produced by reaction of 7mM stock solution of ABTS with 2.45mM potassium persulphate and allowing the mixture to stand in the dark at room temperature for 12-16 h before use. The ABTS•+ solution was diluted with methanol to give an absorbance of 0.70 ± 0.01 at 734nm (Re et al. 1999). Each plant extract (40µL) was serially diluted in a 96-well plate and then allowed to react with 160 µL of the ABTS•+ solution. After 6 min, the absorbance was measured at 714nm (Re et al. 1999). Trolox and ascorbic acid were used as positive controls while the mixture of methanol and ABTS•+ solution was used as the negative control. The blanks were made of methanol and the extracts. Percentage ABTS inhibition was expressed as a percentage following equation 1 above.
The anti-inflammatory activity of H. zeyheri extracts was examined by testing NO inhibition and cytokine expression of both TNF-α and IL-10 in mouse and human macrophage cells.
Nitric oxide production inhibitory assay in mouse RAW 264.7 macrophage cells
The mouse RAW 264.7 macrophage cells were purchased from the American Type Culture Collection (ATCC, Rockville, MD, USA) and cultured in a plastic culture flask in Dulbelcco’s Modified Eagle’s Medium (DMEM) containing L-glutamine (HycloneTM) and supplemented with 10% foetal bovine serum (FBS) (Capricorn Scientific Gmbh, South America) and 1% penicillin/streptomycin/fungizone (PSF) solution at 37°C with 5% CO2. The cells were seeded (10 000 cells per well) in 96 well-microtitre plate and allowed to attach overnight. The cells were then treated simultaneously with LPS alone (control) and extracts at different concentrations. Quercetin and diclofenac were used as the positive controls.
The amount of nitrite produced was determined as described by Dzoyem et al. (2015). Then after 24 hours of incubation at 37°C with 5% CO2, 100µL of cell supernatant from each well were transferred into a new 96-well microtiter plate and the same volume of Griess reagent was added. After 15 min of incubation in the dark at room temperature (25°C), the absorbance was recorded at 550nm on a microtiter plate reader (Epoch Biotek) (Dzoyem et al. 2015). Percentage of NO inhibition was calculated based on the ability of extracts to inhibit nitric oxide production by RAW 264.7 macrophage cells compared with the control (cells treated with LPS alone without samples).
Determination of cytokine expression in human U937 macrophage cells
To prepare the cells, the method described by Passmore et al. (2001) with minor modifications was used. The U937 macrophage cells from the American Type Culture Collection (ATCC, Rockville, MD, USA) were maintained in a humidified atmosphere at 37°C with 5% CO2 in Roswell Park Memorial Institute (RPMI-1640) medium containing L-glutamine (Lonza, SA) and supplemented with 10% foetal bovine serum (FBS) (Capricorn Scientific Gmbh, South America) and 1% penicillin/streptomycin/fungizone (PSF) solution. The cells were seeded (500 000 cells per well) in a 6-well microtitre plate and treated with LPS alone (control) and extracts at different concentrations (Passmore et al. 2001). Quercetin, a flavonoid found in plants with known anti-inflammatory properties and diclofenac were used as the positive controls.
Determination of the cell viability
The cell viability was determined in order to establish whether the inhibition of NO production, inhibition of TNF-α and expression of IL-10 by extracts was not due to their cytotoxic effects. The cytotoxicity of crude extracts was determined using the 3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay as described by Mosmann (1983). After each of the assays, the culture medium was removed from the plates and after washing with 200µL of phosphate buffered saline (PBS), 200µL of fresh culture medium and 30µL of MTT solution (5mg/mL) were added to all wells and the plates were incubated at 37°C with 5% CO2 for 4 h. After incubation, the culture medium was carefully aspirated using a suction pump (Integra, USA), and 50µL of dimethylsulphoxide (DMSO) was added to all wells. The absorbance was read using a microplate reader (Biotek Synergy, USA) at a wavelength of 570nm and a reference wavelength of 630nm. The percentage of cell viability was calculated by comparing the absorbance of the samples to the negative control (cells treated only with LPS considered as 100% viability) (Mossman 1983).
Determination of inhibitory concentration (IC50) and statistical analysis
All experiments were carried out in triplicate and results are presented as mean ± standard deviation. The IC50 value of tested samples, which represent the concentration of the sample required to inhibit 50% of activity compared to the negative control, was determined by using a non-linear regression curve of percentage of inhibition against the logarithm of concentrations. The extracts with lower IC50 values show high activity of the extract or compound. A one-way analysis of variance (ANOVA) was used to test for differences in the measured variables between extracts. All statistical calculations were conducted using R version 3.5.3 with an accepted significance of p < 0.05 in all tests.