All Chemicals for the following assays were purchased from were purchased from Sigma-Aldrich Chemicals, (Steinheim, Germany).
Xanthine oxidase assay
The chemicals that were used in this assay were allopurinol, hypoxanthine, xanthine oxidase from bovine milk and the reagents namely potassium dihydrogen phosphate, potassium hydroxide, ethylene diamine tetraacetic acid disodium salt (EDTANa2), dimethyl sulfoxide (DMSO), nitroblue tetrazolium (NBT), sodium hydroxide and hydrochloric acid.
Nitric oxide assay
The following were used in the nitric oxide assay: RAW 264.7 cell line, trypsin, Trypan Blue dye, menadione, hydrogen peroxide, lipolysaccharide (LPS), Dulbecco’s modified eagle medium (DMEM) supplemented with foetal bovine serum, antibiotics, indomethacin, iodine, potassium iodide, menadione, carbon tetrachloride, hydrogen, and Griess reagent (1 % sulphanilamide and 0.1 % N, (1-N-naphyl) ethylenediamine in 2.5 % phosphoric acid).
The following reagents were used in the antiproliferative assay: Jurkat cells E 6.1 human leukemic T cell lymphocytes (ECACC, Sigma-Aldrich, Dorset, England), Rosewell Park Memorial Institute (RPMI) 1640 media, foetal bovine serum, antibiotics, penicillin, neomycin, streptomycin solution, Hanks Buffered Saline Solution, 1-chloro-2, 4-dinitrobenzene (CDNB), reduced glutathione (GSH), and cisplatin.
Parinari curatellifolia fresh leaves were collected from Centenary (Latitute 16°48’00”S, Longitude 31°07’00”E and elevation above sea level is 1156 m) in Mashonaland Central Province of Zimbabwe in the summer period of February 2012. The plant samples’ identity were authenticated by Mr Christopher Chapano, a taxonomist at the National Herbarium located at the Harare Botanical Gardens, Harare, Zimbabwe. Voucher specimen (C6E7) were made and stored in the Biomolecular Interactions Analyses Laboratory at the Department of Biochemistry, University of Zimbabwe, and Harare, Zimbabwe. The leaves were washed with distilled water and then dried in an oven at 40 ̊C. The leaf powder was prepared using a blender (Philips Co., Shanghai, China). The leaf powder was stored in closed containers at room temperature.
Preparation of plant extracts
The dried P. curatellifolia leaf powder was weighed using the KERNEG balance (Kern and Son GmbH, D -72336 Balingen, Germany) and the following masses of 10 g, 10 g, 15 g and 15 g were mixed with the solvents methanol, water, acetone and ethanol respectively. A volume of 150 mls of each solvent was added to the leaf powder in a 1000 ml beaker. The beakers were placed on a magnetic stirrer and left stirring for 2 h. The solutions were filtered using Whatman No 1 filter paper (Sigma-Aldrich Chemicals, Steinheim, Germany) or transferred into syringe and filtered into a small glass vial. The sterile suspension was filtered again using 0.45 μM Millipore® sterile filter (Sigma-Aldrich, Taufkirchen, Germany) into a labelled small glass vial. The filtrate was evaporated to dryness, collected and stored at 4 oC until when required.
Xanthine oxidase assay
Xanthine oxidase activity assay used was adapted from Bergmeyer et al.  and with slight modifications. Initial screening for the effects of the water, methanol, acetone and ethanol extracts was done on the enzyme and the amount of uric acid produced was quantified spectrophotometrically at 295 nm using a UNICOR, UV/VIS 2800 spectrophotometer (United Products and Instruments Inc., U.S.A). The absorbance values obtained were proportional to the production of uric acid. A unit of activity for xanthine oxidase is that forming one micromole of urate per minute at 25 °C. The activity in the negative control that indicated 100 % enzyme activity was also determined for reactions without the extract. Appropriate volumes and concentrations of reactants: potassium phosphate buffer 50 mM pH 7.4, xanthine oxidase 0.72 U/ml, varying concentrations of the extract, and hypoxanthine 0.15 mM were added into a 1.5 ml quartz cuvette with an appropriate negative control. For the initial screening for inhibitory activity, five extract concentrations of 3.9, 15.6, 63,125 and 250 μg/ml were tested for their effects and the negative control contained no extract. For each of the concentration, the effect on activity was determined in quadruplicate. Superoxide anions are generated by both the hypoxanthine-xanthine oxidase and the xanthine-xanthine oxidase reactions. The superoxide anion radicals produced are detected by coupling them to reduction of nitroblue tetrazolium (NBT). A deep blue colour is produced when the superoxide react with NBT. Thus, colour intensity decreased with a decrease in xanthine oxidase activity. The IC50 determination method was adapted from Wang et al., . Reagents were prepared in 50 mM potassium phosphate buffer of pH 7.4. The reagent mixture contained 20 μL of 15 mM EDTANa2 with pH 7.4, 50 μL of 0.6 mM NBT, 30 μL of 0.5 mM hypoxanthine, 50 μL of xanthine oxidase solution (0.06 U/ml), and 150 μL of various concentrations of plant extract or 150 μL of potassium phosphate buffer (as the control). For the kinetic reactions, a volume of 150 μL of three selected concentrations (0, 0.25, 0.5, and 1.0) μg/ml of allopurinol were added to the wells of the 96 microwell plate. For the extracts concentrations of 0, 1, 2, and 3 μg/ml were used. A volume of 150 μL of potassium phosphate buffer was then added followed by 20 μL of 15 mM EDTANa2, 30 μL of twelve different concentrations (0.01, 0.02, 0.03, 0.04, 0.05, 0.075, 0.1, 0.125, 0.150, 0.175, 0.2, 0.25) μg/ml of 0.5 mM hypoxanthine, 50 μL of 0.6 m nitroblue tetrazolium (NBT). For the methanol extract, the range of the concentration for hypoxanthine was from 0.01 to 0.3 μg/ml. The final concentrations of the other reagents in each well was 0.01 U/ml of xanthine oxidase, 0.1 mM of NBT and 1 mM of EDTANa2. The reaction was initiated by initiated by adding 50 μL of xanthine oxidase solution (0.06 U/ml) at 25 oC, and absorbance values were measured every 30 s for 10 min at 550 nm using the Spectra Max Microplate spectrophotometer (Molecular Devices, Sunnyvale, U.S.A).
Determination of kinetic constants for XO
Determination of kinetic constants for XO was carried out by the method of Wang et al.,  with some modifications. All the reagents were dissolved in the 50 mM potassium phosphate buffer pH 7.4. A volume of 150 μL of three selected concentrations (0, 0.25, 0.5, and 1.0) μg/ml of allopurinol were added to the wells of the 96 microwell plate. A volume of 150 μL of potassium phosphate buffer (as the control), was added followed by 20 μL of 15 mM EDTANa2, 30 μL of twelve different concentrations (0.01, 0.02, 0.03, 0.04, 0.05, 0.075, 0.1, 0.125, 0.150, 0.175, 0.2, 0.25) μg/ml of 0.5 mM hypoxanthine, and 50 μL of 0.6 m nitroblue tetrazolium (NBT). The reaction was initiated by adding 50 μL of xanthine oxidase solution (0.06 U/ml, Sigma-Aldrich). The 96-microwell plate was incubated at 25 oC in a shaking incubator (Jitterbug, 130000, Boekel Industries, Philadelphia, USA), and absorbance measured at 550 nm, readings were taken every 30 s for a period of 10 min using the Spectra Max Microplate spectrophotometer (Sunnyvale, U.S.A). The final concentrations of reagents in each well was 0.01 U/ml of xanthine oxidase, 0.1 mM of NBT, 0.05 mM of hypoxanthine and 1 mM of EDTANa2.
Growth of RAW 264.7
Macrophage cell lines such as the RAW 264.7 cell line can be activated by external triggers to produce nitric oxide in vitro. A 1 ml aliquot of RAW cells was taken from vial and added to 10 ml of DMEM media and incubated at 37 °C and 5 % CO2. The spent media was decanted and fresh media was added and the vial was incubated at 37 ̊oC and 5 % CO2 in Shell Lab® CO2 incubator (Sheldon manufacturing Inc., Cornelius, USA). The confluent cells underwent trypsinisation in order to conduct the cell count with the Trypan blue® exclusion assay. A 200 μL aliquot of the trypsinised cells was mixed with 100 μL of Trypan blue®. The number of cells was determined using a haemocytometer. Viable cells would exclude the dye while dead cells would take up the dye appearing blue.
Determination of the effect of Parinari curatellifolia ethanol leaf extract on nitric oxide production in menadione and hydrogen peroxide-activated RAW 264.7 cells
The effects of P. curatellifolia on nitric oxide production in RAW 264.7 cells after exposure to redox-active compounds menadione and hydrogen peroxide were investigated. The total number of RAW cells was enumerated as above and diluted to obtain a final cell concentration of 2 × 10 5 cells/ml in the assay well of a 12-well plate. A volume of 1.9 ml of DMEM was dispensed into a microplate well and 1 ml of the cell suspension was added and mixed by gentle flushing. A 100 μL aliquot of the test compound was added to a final concentration of a 100 μM and this was followed by 25 μg/ml of P. curatellifolia the ethanol leaf extract. The concentration of 25 μg/ml was chosen as an appropriate non-lethal dose after carrying out a dose-dependent (15.6–250 μg/ml) -study of the effects of the extract. The plate was incubated at 37 oC and 5 % CO2 in a Shell Lab® CO2 incubator (Sheldon manufacturing Inc., Cornelius, USA) for 24 h. The contents of each well was centrifuged at 1500 rpm in a PLC-02® benchtop centrifuge (Gemmy Industrial Corp., Taipei, Taiwan) for 10 mins. The cell free supernatants were measured by the nitrite quantification assay. The effect of the ethanol extract on the inhibition of the production of nitric oxide was determined by quantifying the amount of nitrites in the samples.
Determination of the effect of Parinari curatellifolia on nitric oxide production in lipopolysaccharide -activated RAW 264.7 cells
Cells such as macrophages can express iNOS, the enzyme which is responsible for the production of large amounts of NO when stimulated by antigens such as the Escherichia coli endotoxin, lipopolysaccharide (LPS) . The total number of RAW cells counted were diluted to a final cell concentration of 2 × 105 cells/ml in the assay well of a twelve well plate. A 1.87 ml volume of DMEM was placed in a well and a 1 ml aliquot of the cell suspension was added and mixed. LPS was added to a final concentration of 100 ng/ml in the appropriate wells. The plant water extract at 25 μg/ml was added to the appropriate wells alone or in combination with LPS. The control wells contained media and cells only. The reaction plate was incubated at 37 °C and 5 % CO2 in a Shell Lab® CO2 incubator (Sheldon manufacturing Inc., Cornelius, USA) for 24 h or 48 h. The contents of the wells were centrifuged at 1500 rpm in a PLC-02® benchtop centrifuge (Gemmy Industrial Corp., Taipei, Taiwan) for 10 mins. The cell free supernatants were measured for the amount of NO produced by each experiment using the nitrite quantification assay described below.
Quantification of nitric oxide
NO is converted to nitrite ions in the presence of oxygen. The principle of the assay is that the nitrite ions react with sulphanilamide in acidic conditions to a diazonium salt that in turn reacts with naphylethyldiamine. The reaction produces a pink azo product that absorbs maximally at 540 nm. A standard curve was prepared by two-fold serially diluting 100 μM solution of sodium nitrite in a microplate to a final concentration range of 20 to 1.25 μM. The contents of each sample well were centrifuged at 1500 rpm for 10 min. The cell free supernatants of the test samples were added to wells of the microplate. An equal volume of Griess reagent (1 % sulphanilamide and 0.1 % N, (1-N-naphyl) ethylnediamine in 2.5 % phosphoric acid) was added to the standard wells and test solutions and the plate was incubated for 10 min in the dark at room temperature. The reaction and standard curve samples were read in a Spectramax Plus® UV–Vis microplate spectrophotometer (Molecular Devices Inc., California, USA) at 540 nm.
The effect of glutathione on P. curatellifolia and cisplatin cytotoxicity
P. curatellifolia is used traditionally in the treatment of pain and fever and due to the link between cancer and inflammation, the anti-cancer effects of this plant were also determined on Jurkat cells, a human leukemic cell line. Jurkat cells E 6.1 human leukemic T cell lymphocytes (ECACC, Sigma-Aldrich, Dorset, England) were maintained in RPMI 1640 media supplemented with 10 % heat inactivated foetal bovine serum, 1 % penicillin, neomycin, streptomycin. Cells were grown at 37 °C in 5 % CO2 in a Shel Lab incubator (Sheldon MFG.INC, USA). Jurkat-T cells (1 × 105cells/ml) were seeded in 12-well plates and incubated in a CO2 incubator for 72 h. The cells were pre-treated with cisplatin (0–2 μg/ml) and P. curatellifolia ethanol extract (0–100 μg/ml). A total amount of 20 μl of the extract was used for the extract as well as cisplatin and each concentration was added in duplicate. The cells were counted on a haemocytometer after every 24 h. The effect of glutathione on P. curatellifolia and cisplatin cytotoxicity was also investigated. Jurkat T cells (1 × 105) were seeded in 12 well plates and incubated in a CO2 incubator for 72 h. The cells were pre-treated with 50 μg/ml P. curatellifolia and 0.5 μg/ml cisplatin. The cells were pre-treated with the extract and GSH combined and cisplatin and GSH combined. Cells were also incubated with glutamate an inhibitor of glutathione and extract combined and cisplatin and glutamate combined. A control which had cells in RPMI media was also set up. The experiment was carried out in quadruplicate.
Jurkat cells (1 × 105 cells/ml) were pre-treated with cisplatin (0–2 μg/ml), cisplatin (0–2 μg/ml) and 50 μg/ml P. curatellifolia maintained constant and cisplatin (0–2 μg/ml) and 12 μg/ml ethacrynic acid maintained constant. The cells were incubated at 37 °C for 72 h and then counted on a haemocytometer. Jurkat cells were collected by centrifugation at 2000 rpm for 5 min in a Hettich Rotofix 32 Centrifuge, (Tuttingen, Germany) and the supernatant was discarded. The cells were washed with 5 ml phosphate buffered saline and centrifugation was carried out at 2000 rpm for 5 minutes. The cells were lysed with 100 μl of lysis buffer (10 mM Tris (pH 7.4), 5 mM EDTA, 0.2 % Triton X-100) and 10 μl of 1 mg/ml proteinase K. The cells were incubated overnight at 56 °C in a water bath. A total of 4 μl RNA-ase (100 μg/ml) was added and the cells were further incubated at 37 °C for 1 h. Sodium chloride (1.5 M) was added as 1/10 the volume of the total volume and the tube was inverted several times. The tubes were then centrifuged at 12 000 rpm and the supernatant was retained into clean tubes. A 2 x volume of ice cold isopropyl alcohol was added to the DNA solution and the tubes inverted once more. The tubes were placed in a refrigerator for 1 h at -80 °C. The tubes were then centrifuged at 12 000 rpm for 15 min and the supernatant was discarded. The pellets were then washed with 70 % ethanol to remove any remaining contaminants. The ethanol was allowed to evaporate to leave the DNA. The DNA was re-suspended in TE buffer (10 mM Tris–HCl (pH 7.4) and 0.5 mM EDTA). The DNA was subjected to electrophoresis on a 1.5 % agarose gel at 100 V for 1 h.
Statistically significant differences between the mean of the controls and the tests were analysed using one way ANOVA with Dunnett’s multiple comparison post-test. Enzyme kinetics was analysed with nonlinear regression and allosteric sigmoidal using GraphPad Prism5 (Version 5.03 GraphPad Software Inc. San Diego, California U.S.A).