Chemicals
Naphthalene ethylenediamine dihydrochloride, trichloroacetic acid (TCA), ammonium molybdate, thiobarbituric acid (TBA), orthophosphoric acid, sodium carbonate, sodium acetate, chloroform, concentrated sulfuric acid, trisodium chloride, hexane, acetic acid, sodium phosphate monobasic, potassium phosphate dibasic salt, sodium orthophosphate, sodium nitroprusside, sulfanilamide, sodium dodecyl sulfate, DPPH (1,1 diphenyl-2-picrylhydrazyl), ferrous sulfate. All chemicals and reagents used for this study are of analytical grade and are purchased from Merck Millipore and Sigma-Aldrich Inc. St Louis, USA.
Plant material
The bark and fruits of C. albidum were harvested from Owena-Ijesha community farm, Osun State, Nigeria. They were then identified and assigned specimen voucher number FHI 110105 and deposited at the Forest Research Institute Nigeria (FRIN). The bark and seeds of C. albidum were air-dried for 14 days at room temperature and pulverized.
Preparation of Chrysophyllum albidum extracts
The extraction of C. albidum bark and seeds was done with two solvents (99.8% acetone and 99.8% ethanol) using two methods: maceration and sonication. The extracts were prepared as follows: powdered bark (5 g) was macerated in 100 ml (99.8%) of acetone for 30 min with constant shaking, and the solvent was evaporated to dryness to yield the dried extract labeled CABAM. Another set of powdered bark (10 g) was soaked in 200 ml acetone (99.8%) and subjected to sonication (Model: Sonicator (XUB18UK, Grant) for 30 min to yield the extract labeled CABAS. The powdered seeds (10 g) were soaked in 200 ml acetone (99.8%) and subjected to sonication (Model: Sonicator (XUB18UK, Grant) for 30-min labeled CASAS 30. A separate set of powdered bark (10 g) and powdered seeds (10 g) was soaked in ethanol (200 ml) and subjected to sonication (Model: Sonicator (XUB18UK, Grant) for 30 min, respectively, and the extracts were labeled CABES and CASES, respectively.
Phytochemical screening of the extracts
The extracts were tested for the presence of saponins, steroids, and terpenoids as described by Deepti et al. (2012), Edeoga et al. (2005), and Ghagane et al. (Ghagane et al. 2017); tannins, phlobatannins, and cardiac glycosides (Salkowski’s test) according to the method described by Deepti et al. (2012), Ghagane et al. (2017), and Evans and Trease (2002); and flavonoids as described by, Deepti et al. (2012), Ghagane et al. (2017), and Sofowora (1993).
Evaluation of antioxidant property of extracts
Determination of the total phenolic content of extracts
The total phenolic present in each extract was evaluated by a method previously described by Ghagane et al. (Ghagane et al. 2017), Muhammad et al. (2021), and Singleton et al. (Singleton et al. 1999). In brief, 1 mg/ml of crude extract (200 µL) was mixed with Folin-Ciocalteu reagent (0.5 mL), followed by 20% (w/v) sodium carbonate (2 mL) after 3 min. The mixture was kept in the dark for 1 h and the absorbance was read at 650 nm. A calibration curve was prepared for the estimation of the total phenolic content in each sample using gallic acid. The results were expressed as milligram of gallic acid equivalent per gram dry weight of the extract.
Determination of the total flavonoid content of extracts
The aluminum chloride method previously described by Ghagane et al. (2017), Muhammad et al. (2021), and Chang et al. (2002) was used to determine the total flavonoid contents of the extracts. Briefly, 50 µL of crude extract (1 mg/mL) was mixed with methanol to make 1 ml of the mixture. The mixture was further mixed with 4 mL of distilled water and incubated for 5 min. Then, 0.3 mL of AlCl3 (10%) and 0.3 mL of NaNO2 (5%) were added to the mixture and allowed to settle. After 6 min, 1 M NaOH (2 mL) was added to the mixture and incubated for 15 min. The absorbance of each mixture was taken at 510 nm and the total flavonoid content was estimated from a quercetin calibration curve. The result was expressed as milligram quercetin equivalent per gram dry weight of the extract.
Determination of the total antioxidant capacity of the extracts
The total antioxidant activity of each extract was calculated by the method described by Ghagane et al. (2017), Muhammad et al. (2021), and Prieto et al. (1999). 0.1 mL of sample (100 μg) solution was combined with 1 mL of reagent (0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate). It was then incubated at 95 °C in a water bath for 90 min. The absorbance was read at 695 nm against blank in a spectrophotometer, after cooling to room temperature. An equal volume of solvent used to dissolve the sample was incubated with 1 mL of reagent solution under the same conditions as the samples, which served the blank. The antioxidant capacity of each extract was expressed as equivalents of ascorbic acid.
Evaluation of DPPH radical scavenging activity of the extracts
In this experiment, the DPPH radical scavenging activity was determined according to the method of Ghagane et al. (2017), Muhammad et al. (2021), and Manzocco et al. (1998). 0.0158 g of 1,1-diphenyl-2-picrylhydrazyl (DPPH) was dissolved in 400 ml of methanol. 1 ml of it was added to 1 ml of extracts of various concentrations ranging from 5 µg/ml to 100 µg/ml and allowed to react at room temperature for 30 min. The reaction mixture was vortexed thoroughly and incubated at room temperature in the dark for 30 min. The absorbance of the mixture was read at 517 nm. DPPH radical scavenging activity of extracts and standard was expressed as % scavenging activity. It was calculated using the given formula: % Scavenging activity = ((Ao − A)/Ao) × 100 where Ao is the absorbance of the control and A is the absorbance of the extract. The percentage of DPPH radical scavenging activity was used to acquire the IC50 value of each extract, defined as the concentration of extract necessary to cause 50% inhibition. The smaller the IC50 value, the higher is the antioxidant activity.
Evaluation of nitric oxide scavenging activity of the extracts
In this experiment, the nitric oxide radical scavenging activity was determined according to the method of Marcocci et al. (1994). 2 mL of 10 mM sodium nitroprusside dissolved in 0.5 mL phosphate buffer saline (pH 7.4) was mixed with 0.5 mL of extract at various concentrations (5 µg/ml to 100 µg/ml). The mixture was then incubated at 25 °C. After 150 min of incubation, 0.5 ml of the incubated solution was withdrawn and mixed with 0.5 mL of Griess reagent (2% sulfanilamide in 5% phosphoric acid, and 0.2% naphthyl ethylenediamine dihydrochloride (NEDD)). The mixture was then incubated at room temperature for 30 min and its absorbance was measured at 546 nm. Nitric oxide radical scavenging activity of extracts and standard (ascorbic acid) was expressed as % scavenging activity. It was calculated using the given formula: % Scavenging activity = ((Ao − A)/Ao) × 100 where Ao is the absorbance of the control and A is the absorbance of the extract. The percentage of nitric oxide radical scavenging activity was used to acquire the IC50 value of each extract, defined as the concentration of extract necessary to cause 50% inhibition. The smaller the IC50 value, the higher is the antioxidant activity.
Assay of lipid peroxidation inhibition activity of the extracts
Yolk sample (50 g) was dissolved in 150 ml of chloroform and 1% sulfuric acid in methanol (75 ml). The mixture was mixed in a stoppered tube for 2 h. 50 ml of 5% NaCl solution was added and the required esters were extracted with hexane (3 \(\times\) 30 ml) using Pasteur pipette, and the layer was separated. The hexane aliquot was washed with 2% KHCO3 (40 ml) and dried over anhydrous sodium sulfate. The solution was then filtered to remove the drying agent (Kaźmierska et al. 2005). Lipid peroxidation inhibitory activity of each extract was determined according to the method of Ohkawa et al. (Ohkawa et al. 1979). The tissue (egg yolk lipid extract) was homogenized in 0.1 M buffer pH 7.4 with a Teflon-glass homogenizer. Lipid peroxidation (LPO) in this homogenate was determined by measuring the amounts of malondialdehyde (MDA) produced primarily. Extracts at varying concentrations (5 µg/ml to 100 µg/ml) were added to 100 μl of (15 mM) ferrous sulfate followed by the addition of 3 ml of homogenate. After incubation for 30 min, 0.1 ml of this reaction mixture was mixed with 1.5 ml of 10% TCA and incubated for 10 min. 1.5 ml of 0.67% TBA (in 50% acetic acid) was added and placed in a boiling water bath for 30 min. After centrifugation at 3000 rpm for 10 min, the upper organic layer was taken and its OD (optical density) was measured at 532 nm against an appropriate blank without the sample. The lipid peroxidation inhibitory activity of the extracts and standard (ascorbic acid) was expressed as % inhibition. It was calculated using the following formula: % Inhibition = ((Ao − A)/Ao) × 100 where Ao is the absorbance of the control and A is the absorbance of the extract. The percentage of nitric oxide radical scavenging activity was used to acquire the IC50 value of each extract, defined as the concentration of extract necessary to cause 50% inhibition. The smaller the IC50 value, the higher is the antioxidant activity.
Statistical analysis
All the analyses were run in triplicates. Results were then computed using Microsoft Excel software (Microsoft Corporation, Redmond, WA) and analyzed using appropriate analysis of variance (ANOVA) followed by Duncan’s new multiple range test (DNMRT) and Pearson’s correlation analysis using GraphPad Prism 6.01 (GraphPad Software Inc., CA, USA). The criterion for statistical significance level was set at p < 0.05.