Extraction of the stem bark of Alstonia boonei
The stem bark of A. boonei were peeled from the plant growing in an uncultivated farmland in Ibadan. Samples were authenticated by Mr. Omotayo F.L., Ekiti State University Herbarium and a specimen (UHAE 013) was deposited in the herbarium. The stem bark (100 g) were air-dried at room temperature for five weeks, crushed and soaked in methanol (MeOH) for 72 h with constant shaking. The extract was sieved and concentrated using a rotary evaporator at 40 ºC under reduced pressure.
Purification of the bioactive compound
The MeOH fraction (2g) was partitioned successively using ethylacetate (EtOAc, 100%), EtOAc: MeOH (1:1) and MeOH (100%) using vacuum liquid chromatography. Based on biological activity, using the mitochondrial permeability transition pore opening, the EtOAc-MeOH fraction was further purified using column chromatography starting with 100% chloroform that was stepped by 10% to 100% EtOAc. Thereafter, the polarity of the solvent system was increased by adding MeOH at 5% intervals until 20% MeOH was added. Eight fractions of each were collected for each eluent step. These fractions were pooled based on their thin layer chromatography (TCL) profiles to give fractions A (1-9), B(10-27), C(28-42), D(43-72) and E(73-104). Fraction C was further purified using preparative TLC to yield 2-methyl-3-propylbutane-1,4-diol. The purified compound was subjected to 1H, 13C, COSY, HMBC and HSQC spectroscopic analyses for full identification and characterization.
All animals used for this experiment were handled in accordance with the rules and regulations for management of animals used in research as contained in the Guide for the Care and Use of Laboratory Animals19. Furthermore, this study was approved by the University of Ibadan Animal Use and Care Research Ethics (ACUREC) and an approval number UI-ACUREC/18/0053 was assigned to the study. Furthermore, this study was carried out in compliance with the ARRIVE guidelines and in accordance with relevant guidelines and regulations. The use of this medicinal plant complies with local and national regulations. Spectroscopic data generated on the purification of the natural product used in this study are included in this published article as supplementary information files.
Isolation of mitochondria from rat liver
Mitochondria were isolated from Wistar rat liver according to a previously described method by Johnson and Lardy20. The rats were sacrificed by cervical dislocation, dissected, the liver was removed, rinsed in isolation buffer (210 mM mannitol, 70 mM sucrose, 5 mM HEPES-KOH (pH 7.4) and 1 mM EGTA), and a 10% minced suspension of the liver in isolation buffer was homogenised on ice-cold medium. The homogenate was loaded into a cold centrifuge (Sigma 3-30K, Germany) and spun twice at 2300 rpm for 5 min each to remove cell and nuclear debris and unbroken cells. The supernatant was spun at 13000 rpm for 10 min to sediment the mitochondria. Mitochondria pellets were washed twice with washing buffer (210 mM mannitol, 70 mM sucrose, 5 mM HEPES-KOH (pH7.4) and 0.5% BSA) at 12000rpm for 10 min each time after which, mitochondria were dispensed into aliquots in Eppendorf tubes with suspension buffer (210 mM mannitol, 70 mM sucrose and 5 mM HEPES-KOH (pH7.4)). Mitochondria used for ATPase activity were isolated similarly except sucrose buffer (25 M) was used throughout.
Mitochondrial Protein determination
Mitochondrial protein was determined as previously described using bovine serum albumin (BSA) as the standard21. Mitochondria (10 µl) were pipetted into 990 µl of distilled water and 3 ml of a 100:1:1 solution of 2% Na2CO3, 2% Na-K tartrate and 2% CuSO4.5H2O was added. The mixture was incubated at room temperature for 10 min, after which, a five-fold diluted Folin-Ciocalteau (0.3 ml) was added. This was incubated at room temperature for 30 min, vortexed and the absorbance was read at 750 nm using a spectrophotometer (752N UV-Visible spectrophotometer, China). Mitochondrial protein was quantified from the BSA standard curve.
Mitochondria permeability transition pore opening
The opening of the mitochondrial pore by MPBD was assessed according to the method described by Lapidus and Sokolove22. This method was first used to determine the quality and suitability of the isolated mitochondria for mPT assay as follows: Mitochondria protein (0.4 mg/mL) was incubated in suspension buffer in the presence of rotenone (0.8 µM) for 3.5 min after which, succinate (5 mM sodium succinate) was added and the absorbance was monitored at 540 nm for 12 min at 30 s interval. Furthermore, the pore opening effect of calcium; a standard pore opening inducer was monitored using the same mitochondria protein incubated in suspension buffer in the presence of rotenone for 3 min after which, calcium (3 µM CaCl2) was added. Thirty seconds later, succinate was added to energise the medium and the absorbance was read. Spermine reversal of calcium-induced opening was done by incubating the same mitochondria protein quantity in suspension buffer in the presence of rotenone and 5 mM spermine for 3 min. Thereafter, calcium was added. The reaction medium was energised with succinate 30s later and the absorbance was monitored for 12 min at 30s intervals. Mitochondria whose calcium-induced pore opening is effectively reversed by spermine to the tune of ≥80% were adjudged to be intact, retain their integrity, uncoupled and therefore, suitable for the assay.
To assess the effect of MPBD in the absence of calcium on mPT, mitochondria were incubated in the presence of graded concentrations (10-80µg/ml) of this compound in suspension buffer and rotenone and further energized with succinate. Effect of MPBD on mPT in the presence of calcium follows similar process only that calcium is appropriately added.
Mitochondrial ATPase activity
In triplicate, sucrose (0.25M), KCl (5mM) and Tris-HCl (0.1M) were dispensed into test tubes and the entire volume was made up to 1 ml with distilled water. Adenosine triphosphate (0.01 M) was added to all test tubes except the ‘mitochondria only’ tubes. Mitochondria (0.5 mg/ml) were added to all test tubes except the ‘ATP only’ tubes. Uncoupler (25 µM, 2,4-dinitrophenol) was added to the designated tubes while 10% sodium dodecylsulphate (SDS) was added to the zero time tubes immediately after the addition of mitochondria. Varied concentrations of the test compound (10-80 µg/ml) were added to the test group containing ATP and mitochondria. The volume was made up and the test tubes were transferred to the shaker water bath and they were incubated at 27ºC for 30 min. After incubation, 1 ml of SDS was added to all the tubes except the zero tubes to stop the reaction. Thereafter, 1 ml each of the mixture was transferred into separate test tubes and 1 ml each of 1.25% ammonium molybdate in 6.25% H2SO4 and 9% ascorbate were added successively and the solution was allowed to stand for 30 min. The absorbance of the blue color was read at 660 nm. The concentration of inorganic phosphate was estimated from a 1 mM solution of potassium dihydrogen phosphate, which was treated like the deproteinised mixture23.
Cytochrome c release determination
Cytochrome c release was determination as previously described24. Briefly, mitochondrial protein was pre-incubated in suspension buffer, rotenone and graded concentrations (10-80 µg/mL) of the test compound for 3.5 min, thereafter, succinate was added. For the negative control, mitochondria were pre-incubated in the absence of calcium whilst in the positive control mitochondria were pre-incubated in the presence of calcium. The test compound was similarly treated in the absence of mitochondria and the incubation in each case lasted for 30 min after which the mixtures were spun at 12000 rpm for 10 min. The absorbance of the supernatant was read at 414 nm while the absorbance of the drug control was deducted from that of the test groups. The concentration of cytochrome c release was estimated from the cytochrome c standard curve.
Membrane stabilizing activity
The procedure for the membrane stabilising assay was carried out as previously described25. The 2 ml assay mixture contained 0.5ml hyposaline, 1.0ml of 0.15M sodium phosphate buffer (pH 7.4), varying volumes of isosaline and 0.5ml of bovine erythrocyte suspension. The drug control assay contained the above volumes of buffers and reagents without erythrocyte suspension while the blood control contained all the reagents except the drug.The reaction mixtures were incubated at 56ºC for 30 min in a water bath and cooled to room temperature. Thereafter, mixtures were centrifuged at 3000 rpm for 5min and the absorbance of the supernatant was read at 560 nm against the test blank; the buffered sodium chloride solution served as blank. The percentage membrane stability activities were estimated as follows:
Hypotonic solution-induced hemolysis
Using the hypotonic solution-induced erythrocyte lysis,whole bovine blood was centrifuged and blood cells were washed three times with 154mM NaCl in 10mM sodium phosphate buffer (pH 7.4) through centrifugation for 10 min at 3000rpm. The sample tubes contained 0.50ml erythrocyte suspension mixed with 5mL of hypotonic solution (50 mM NaCl) in 10mM sodium phosphate buffered saline (pH 7.4) containing the compound (2.0mg/ml) or acetyl salicylic acid(0.1mg/ml). The control sample consisted of 0.5ml of erythrocytes mixed with hypotonic buffered saline alone .The mixture was incubated for 10 min at room temperature, centrifuged for 10 min at 3000 rpm and the absorbance of the supernatant was measured at 540nm. Acetyl salicylic acid was used as the positive control.
Representative profiles were used for the determination of mitochondrial permeability transition while other results were presented as mean + SD of triplicate determinations. Data were analyzed using descriptive statistics and one way ANOVA with Tukey multiple comparison post-hoc test to compare groups.