2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) or ABTS and dichlorofluorescin diacetate (DCFHDA) were obtained from Sigma (St. Louis, MO, USA). 6-hydroxy-2,5,7,8-tetramethychroman-2-carboxylic acid (Trolox) and 2,2-diphenlyl-1-picrylhydrazyl (DPPH) were obtained from Fluka, Buchs, Switzerland. Potassium persulfate (K2S2O8), ethylenediaminetetraacetic acid (EDTA), ascorbic acid, 2- deoxy-2-ribose, trichloroacetic acid (TCA), and quercetin were obtained from Sisco Research Laboratories Pvt. Ltd., Mumbai, India. Hydrogen peroxide, Ferrous sulfate, potassium hexacyanoferrate, Folin-Ciocalteu reagent, sodium carbonate (Na2CO3) and butylated hydroxytoluene (BHT) were obtained from Merck, Mumbai, India. Thiobarbituric acid (TBA) was obtained from Loba Chemie, Mumbai, India.
Fresh leaves of Parkia biglobosa were collected in Isua-Akoko, Ondo State, Nigeria. Botanical identification and authentication was carried out by Dr. Ugbogu A.O and Mr. Shasanya O.S at the herbarium of the Forestry Research Institute (FRIN) Ibadan, Oyo state, Nigeria where a voucher specimen (no 109603) was deposited.
Parkia biglobosa Extract (PBE) preparation
The leaves were air-dried for 28 days at room temperature and ground to fine powder using a blender. A 500 g sample of the powdered material was macerated in 1200 mL of a mixture of methanol and water (4:1) for 48 h. This was filtered and concentrated to a small volume to remove the entire methanol using rotary evaporator. The concentrated extract was then lyophilized. The residue was kept at −20 °C for future use. Extract yield was approximately 11%.
Male Wistar rats (±3 months old), weighing between 270 g and 320 g, from the University breeding colony (Animal House Holding, UFSM, Brazil) were kept in cages with free access to foods and water in a room with controlled temperature (22 °C ± 3) and in 12-h light/dark cycle with lights on at 7:00 a.m. The animals were maintained and used in accordance to the guidelines of the Brazilian Association for Laboratory Animal Science.
Preparation of tissue homogenates
Rats were sacrificed by decapitation on the day of experiment and rapidly dissected to harvest the whole brain and liver which were then placed on ice and weighed. Tissues were immediately homogenized in ten volumes of cold (4 °C) Tris–HCl (10 mM, pH 7.4). The homogenate was centrifuged for 10 min at 4000 g to yield a pellet that was discarded and a low-speed supernatant that was used in thiobarbituric acid reactive substances (TBARs) quantification.
In vitro antioxidant/radical scavenging and metal ion chelating activities assays
Total antioxidant activity
Total antioxidant activity was determined by the ABTS test described by Re et al. . The ABTS.+ radical cation was generated by mixing 7 mM ABTS stock solution with 2.45 mM potassium persulfate (final concentration) and incubating for 12–16 h in the dark at room temperature. The absorbance of the ABTS.+ solution was equilibrated to 0.70 (± 0.02) by diluting with distilled water. ABTS.+ solution (1 ml) was mixed with 10 μl of PBE dissolved in distilled water (0, 10, 25, 50, 100 and 150 μg/ml final concentration) or Trolox standard dissolved in deionized water (0, 1, 2.5, 5.0, 7.5, and 10 μg/ml final concentration). The absorbance was measured at 734 nm after 6 min. All experiments were carried out in replicates. The percentage inhibition of absorbance was calculated and plotted as a function of the concentration of standard and sample to determine the Trolox equivalent antioxidant concentration (TEAC). To calculate the TEAC, the straight line gradient of the plot for the sample was divided by that of Trolox (Additional file 1: Figure S1).
DPPH radical scavenging activity of extract
DPPH radical-scavenging activities of P. biglobosa extract and reference compound (Ascorbic acid) were determined as described by Batool et al. . The capacity of extracts to scavenge the lipid-soluble 2, 2- diphenyl-1-picrylhydrazyl (DPPH) radical, which results in the bleaching of the purple colour exhibited by the stable DPPH radical, could be monitored at 517 nm.
Briefly, PBE (0, 10, 25, 50, 100, and 250 μg/ml) or the reference compound, ascorbic acid (0, 10, 20, 30, 40 and 50 μg/ml) was added to an ethanol solution of DPPH (0.03 mM). The mixture was shaken and left to stand at room temperature for 30 min. The absorbance of the resulting solution was measured spectrophotometrically at 517 nm. The radical scavenging activity was calculated as a percentage of DPPH. discolouration.
The Fe3+- reducing power of the extract was determined as described by Oyaizu  with a slight modification. Different concentrations (0.0–200 μg/mL) of the extract (0.5 mL) were mixed with 0.5 mL phosphate buffer (0.2 M, pH 6.6) and 0.5 mL potassium hexacyanoferrate (0.1%), followed by incubation at 50 °C in a water bath for 20 min. After incubation, 0.5 mL of TCA (10%) was added to terminate the reaction. The upper portion of the solution (1 mL) was mixed with 1 mL distilled water, and 0.1 mL FeCl3 solution (1%) was added. The reaction mixture was left for 10 min at room temperature and the absorbance was measured at 700 nm against an appropriate blank solution. All tests were performed three times. A higher absorbance of the reaction mixture indicated greater reducing power. Butylated hydroxytoluene (BHT) was used as a positive control.
The ferrous ion chelating activity of extract was evaluated by a standard method  with minor changes. The reaction was carried out in Tris-HCL buffer (0.1 M, pH 7.5). Briefly, various concentrations (0–200 μg/mL) of plant extract were added to 100 μM ferrous sulfate solution. The reaction mixture was incubated for 30 s, before the addition of 1, 10-Phenanthroline (0.25% w/v). The absorbance was subsequently measured at 510 nm in a spectrophotometer. EDTA was used as a positive control.
Hydroxyl radical scavenging
This was assayed by a standard method . Deoxyribose is degraded by hydroxyl radicals with the release of thiobarbituric acid (TBA) reactive materials. The assay was based on the generation of hydroxyl radical by the Fe2+-H2O2 system (the Fenton reaction) and quantification of the degradation product of 2-deoxyribose by condensation with TBA.
The reaction mixture contained 120 μl of 2-deoxy-2-ribose (3 mM); 80 μl of potassium phosphate buffer (50 mM, pH 7.4); 80 μl of FeSO4 (100 μM); 80 μl of H2O2 (1.0 mM) and 40 μl of PBE (0, 25, 50, 100, 150, and 200 μg/ml) of the test sample and distilled water to make up a final volume of 1 ml. After incubation for 1 h at 37 °C, 0.5 ml of the reaction mixture was added to 1 ml of 2.8% (w/v) TCA, then 1 ml of 1% aqueous TBA was added. The mixture was incubated at 90 °C for 15 min. After cooling, the absorbance was measured at 532 nm against an appropriate blank solution. The percentage inhibition was evaluated by comparing the test and blank solutions.
Evaluation of membrane lipid peroxidation
Quantification of thiobarbituric acid reactive substances (TBARs) production, an index of biological membrane peroxidation, was determined as described by Puntel et al. . Briefly, 20 μL of PBE (50–250 μg/mL) and prooxidant agent (100 μm Fe2+) were added to 100 μL of rat liver or brain tissue homogenate in Tris-HCL buffer (10 mM; pH 7.4). The reaction mixture was incubated at 37 °C in a water bath. Color reaction was developed by adding 200 μL of 8.1% sodium dodecyl sulfate (SDS) to the reaction mixture. This was subsequently followed by the addition of 500 μL of acetic acid/HCl buffer (1.34 M; pH 3.4) and 500 μL 0.6% thiobarbituric acid (TBA). The mixture was incubated at 100 °C for 1 h. TBARs produced were measured at 532 nm and the absorbance was compared with a malondialdehyde (MDA) standard curve.
Angiotensin-converting enzyme (ACE) inhibition assay
The assay was based on the hydrolysis of N-hippuryl-His-Leu hydrate (HHL) by the angiotensin-converting enzyme as described by Cushman and Cheung . Briefly, the enzyme source was prepared with freshly removed rat lung. The tissue was homogenised in cold 125 mM Tris buffer, pH 8.3 (1/10, w/v) and centrifuged at 4 °C for 10 min at 4000 g to yield a low-speed supernatant. The reaction mixture contains 40 μL Tris-HCl buffer (125 mM, pH 8.3), enzyme source (50 μL) and 10 μL extracts/drug (PBE- 25, 50, 100 μg/mL; ramipril- 0.04, 0.2, 0.4 μg/mL). This was incubated at 37 °C for 15 min. Thereafter, ACE substrate, HHL (8.3 mM; 150 μl) was added and further incubated for 30 min at the same temperature in a shaker. The enzymic reactions were terminated by addition of 1 ml of 1 M HCI. The hippuric acid formed by the action of the angiotensin-converting enzyme on HHL was extracted from the acidified solution into l-2 ml of ethyl acetate by vortex mixing for 15 s. After centrifugation at 3000 g for 5 min, an l ml aliquot of each ethyl acetate layer was transferred into a clean tube. The ethyl acetate fractions were evaporated by heating at 120 °C for 30 min in a Temp-Blok module heater. The hippuric acid was re-dissolved in 1 ml distilled water and the amount formed was determined from its absorbance at 228 nm wavelength.
Isolation of hepatic mitochondria
Liver mitochondria were isolated as previously described . Wistar rats were killed by decapitation and the liver tissues were rapidly removed and placed on ice-cold isolation buffer containing 225 mM mannitol, 75 mM sucrose, 1 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 0.1% bovine serum albumin (BSA; free fatty acid) and 10 mM HEPES pH 7.2. The tissues were then homogenized and the resulting suspension centrifuged for 7 min at 2,000Xg. Next, the supernatant was centrifuged for 10 min at 12,000Xg. The pellet was re-suspended in isolation buffer II containing 225 mM mannitol, 75 mM sucrose, 1 mM EGTA and 10 mM HEPES pH 7.2 and centrifuged at 12,000Xg for 10 min. Finally, the last supernatant was discarded, and the pellet was re-suspended and maintained in buffer III (sucrose 100 mM, KCl 65 mM, K+-HEPES 10 mM and EGTA 50 μM pH 7.2) to a protein concentration of 0.5 mg/mL for subsequent analyses. Protein concentration was measured by the method described by Lowry et al.  using bovine serum albumin (BSA) as standard.
Measurement of mitochondrial membrane potential (∆ψm)
Mitochondrial membrane potential was estimated by fluorescence changes of safranine recorded by spectrofluorimeter . The cuvette inside the spectrofluorimeter contains 3 mL of buffer III to which an aliquot (30 mL) of the isolated mitochondria (approximately 500 mg protein) was added. The reaction was started by the addition of safranine (67 mM) and succinate (1.5 M) added after 10 s. The fluorescence was monitored for 200 s after which 10 μL of extract/drug (PBE: 25, 50, 100 μg/mL; catechin: 1, 5, 10 μg/mL) or distilled water (for control) was added and allowed for additional 150 s. Finally 2,4-dinitrophenol, DNP (100 mM, 30 μL) was added to uncouple oxidative phosphorylation and inhibit adenosine triphosphate production. The change in fluorescence was recorded by a RF-5301 Shimadzu spectrofluorimeter (Kyoto, Japan) operating at excitation and emission wavelengths of 495 nm and 586 nm, respectively, with slit widths of 3 nm. The potential difference (∆Ψm) was obtained by the difference between the fluorescence intensity prior to and after DNP addition.
Evaluation of reactive species (RS) formation with DCH (dichlorofluorescein-reactive species (DCH-RS)
RS levels were measured using the oxidant sensing fluorescent probe, 2′,7′-dichlorofluorescein diacetate (DCHF–DA) . The oxidation of DCHF–DA to fluorescent dichlorofluorescein (DCF) was determined at 488 nm for excitation and 525 nm for emission. An aliquot of 5 μL (50 μg/protein) of the homogenate of the isolated mitochondria was added to 3 mL of buffer III (containing 5 mM succinate). The reaction medium was exposed to PBE (25, 50, 100 μg/mL) or catechin (1, 5, 10 μg/mL) with or without Ca2+ (80 μM) or SNP (150 μM). After 10 s, DCHF–DA (10 μM, in absolute ethanol) was added to the mixture and the fluorescence intensity from DCF was measured for 300 s. Values of mitochondrial membrane potential (∆ψm) were expressed as percent of control.
Results calculated from triplicate data were expressed as means ± standard error of means (SEM). With the exception of data on mitochondrial ROS production which was analysed using two-way ANOVA followed by Bonferroni post test, data were analyzed using one-way analysis of variance followed by Neuman-Keuls comparison of means. The significance level was set at p < 0.05. Statistical analysis, graphing and EC50/IC50 (median effective concentration/median inhibitory concentration) determination were done using Graph Pad Prism (ver.5.0a).