Plant material
The leaves and barks of M. myristica were collected at the Kala Mountain in the Center region of Cameroon. They were authenticated by M. NANA, a botanist of the National Herbarium of Cameroon, in comparison to the voucher specimens (27690/SFR/CAM).
Preparation of plant extracts
The collected leaves and barks were dried at ambient temperature and crushed. The powders were then macerated in the ratio of 1:10 (w/v) for 48 h in ethanol for the ethanolic extract and in a mixture of water + ethanol (30/70); pH = 3 for the hydro-ethanolic extract. The mixtures were then filtered using a Buchner funnel and Whatman No 1 filter paper. This process was repeated once on the residue. The filtrate was concentrated using a rotavapor and the solution was dried in the oven at 55°C for two days. Each crude extract obtained was labelled using the following codes: AFE: M. myristica (leaves) ethanolic extract; AEH: M. myristica (Barks) hydroethanolic extract; AEE: M. myristica (Barks) ethanolic extract; AFH: M. myristica (leaves) hydroethanolic extract. The different samples were then kept at 4°C. Prior to the experimentation, the solutions of the four plant extracts were dissolved using ethanol different dilutions (25, 50, 75, 150, 300 μg/mL) of each.
Animals
Male albino Wistar rats weighing 200–250 g were used in this study. The rats were maintained at room temperature under the lab conditions and were fed with standard diet and water ad libitum. Livers were collected after decapitation of the rats under mild ether anesthesia from overnight fasted rats. This study was carried out with approval from the animal Ethics Committee of university of Yaoundé I.
Determination of the free radical scavenging potential of the samples
Scavenging activity of DPPH radical
The DPPH assay measures the free radical scavenging capacity of the extracts as described previously [40]. Three milliliters of each of the diluted extracts were put in the test tube and 1 mL of a methanol solution of DPPH (0.1 mM) was added. The mixture was kept in the dark at room temperature for 30 min and absorbance was measured at 517 nm against a blank. The same procedure was used for the vitamin C used as standard. The following equation was used to determine the percentage of the radical scavenging activity of each extract.
$$ Scavenging\; effect\left(\%\right)=100\times \left({A}_0-{A}_S\right)/{A}_0 $$
where Ao is the absorbance of the blank and As the absorbance of the sample.
Scavenging effect of the ABTS+ radical
The ABTS assay was based on a previously described method [41] with slight modifications. ABTS radical cation (ABTS+) was produced by the reaction of a 7 mM ABTS solution with potassium persulphate (2.45 mM). The ABTS+ solution was diluted with ethanol to an absorbance of 0.70 ± 0.05 at 734 nm. The mixture was stored in the dark at room temperature for 12 h before used. After addition of 25 μL of extract sample or vitamin C used as standard to 2 mL of diluted ABTS+ solution, absorbance was measured at 734 nm after exactly 6 min. The decrease in absorption was used for calculating scavenging effect values. The following equation was used to determine the percentage of the radical scavenging activity of each extract.
$$ Scavenging\; effect\left(\%\right)=100\times \left({A}_0-{A}_S\right)/{A}_0 $$
where Ao is the absorbance of the blank; As is the absorbance of the sample.
Nitric oxide scavenging activity
Nitric oxide scavenging activity was determined according previous authors [42]. The reaction mixture contained 2 mL of sodium nitroprusside (10 mM) in 0.5 mL phosphate buffer (0.5 M; pH 7.4). Various concentrations (25, 50, 75, 150, 300 μg/mL) of the extracts (0.5 mL) were added in a final volume of 3 mL. After incubation for 60 min at 37°C, Griess reagent [α-napthyl-ethylenediamine (0.1%) and sulphanilic acid (1%) in H3PO4 (5%)] was added. The pink chromophore generated during diazotization of nitrite ions with sulphanilamide and subsequent coupling with α-napthyl-ethylenediamine was measured spectrophotometrically at 540 nm. Ascorbic acid was used as a positive control. The scavenging ability (%) of the nitric oxide was calculated using the formula:
$$ Scavenging\; effect\left(\%\right)=100\times \left({A}_0-{A}_S\right)/{A}_0 $$
where Ao is the absorbance of the blank and As the absorbance of the sample.
Hydroxyl radical scavenging activity
The scavenging activity of the extracts on hydroxyl radical was measured according to a previously described method [43]. To 1.5 mL of each diluted extract, 60 μL of FeCl3 (1 mM), 90 μL of 1,10-phenanthroline (1 mM), 2.4 mL of phosphate buffer (0.2 M; pH 7.8) and 150 μL of H2O2 (0.17 M) were added respectively. The mixture was then homogenized using a vortex and incubated at room temperature for 5 min. The absorbance was read at 560 nm against the blank. The percentage of the radical scavenging activity of each extract was calculated from the equation below:
$$ Scavenging\; effect\left(\%\right)=100\times \left({A}_0-{A}_S\right)/{A}_0 $$
where Ao is the absorbance of the blank and As the absorbance of the sample.
Determination of the total antioxidant potential of the different samples
Total antioxidant activity by Ferric Reducing Antioxidant Power assay (FRAP)
The FRAP was determined using a previously described method [44] with slight modifications. The fresh FRAP reagent consisted of 500 mL of acetate buffer (300 mM; pH 3.6), 50 mL of 2,4,6- Tri (2-pyridyl)-s-triazin (TPTZ) (10 mM), and 50 mL of FeCl3•6H2O (50 mM). The colorimetric measurement was performed at 593 nm and the measurement was monitored up to 12 min on 75 μL of each extract and 2 mL of FRAP reagent. The vitamin C was used to draw a standard curve and the Butylated Hydroxy Toluene (BHT) was used for the comparison. The results were expressed as mg equivalent vitamin C/g of dried extract (mg eq VitC/g DE).
Phosphomolybdenum Antioxidative Power (PAP)
The total antioxidant activity of the extracts was evaluated by green phosphomolybdenum complex [45]. An aliquot of 10 μL of the extract solution was mixed with 1 mL of reagent solution (0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate) in a micro centrifuge tube. The tubes were incubated in a dry thermal bath at 95°C for 90 min. After cooling, the absorbance of the mixture was measured at 695 nm against a blank. The vitamin C was used as reference to draw the standard curve and BHT was used for the comparison. The reducing capacities of the analysed extracts were expressed as mg of ascorbic acid equivalents/g of dried extract (mg eq AS/g DE).
Reducing power assay
The reducing power of the extracts was determined by a method described by Oyaizu [46]. Different concentrations of extracts in 1 mL of distilled water were mixed with 2.5 mL of phosphate buffer (0.2 M, pH 6.6) and 2.5 mL of potassium ferrocyanide (1%). The mixtures were incubated at 50°C for 20 min. Aliquots 2.5 mL of trichloroacetic acid (10%) were added to the mixtures and centrifuged at 3000 rpm for 10 min. The supernatant of the solution (2.5 mL) was mixed with 2.5 mL of distilled water and 0.5 mL of FeCl3 (0.1%). The absorbance was measured at 700 nm.
Determination of the phenolic content of the extracts
Total phenol determination
The total phenol content was determined by the Folin–Ciocalteu method [47]. The reaction mixture contained 200 μL of extract, 800 μL of freshly prepared diluted Folin Ciocalteu reagent and 2 ml of sodium carbonate (7.5%). The final mixture was diluted to 7 mL with deionized water and kept in the dark at ambient conditions for 2 h to complete the reaction. The absorbance was measured at 765 nm. Caffeic acid was used as standard and the results were expressed as mg caffeic acid/g dried extract (mg CA/g DE).
Determination of total flavonoid content
Total flavonoid content was determined using aluminium chloride (AlCl3) according to a known method [48]using quercetin as a standard. A volume of 0.1 mL of spice extract was added to 0.3 mL distilled water followed by 0.03 mL of NaNO2 (5%). After 5 min at 25°C, 0.03 mL of AlCl3 (10%) was added. After a further 5 min, the reaction mixture was mixed with 0.2 mL of 1 mM NaOH. Finally, the reaction mixture was diluted to 1 mL with water and the absorbance was measured at 510 nm. The results were expressed as quercetin equivalent mg/g of dried extract (QE/g dried ext).
Determination of total flavonols
Total flavonols in the plant extracts were estimated using a known method [49] with modifications. To 2.0 mL of sample, 2.0 mL of 2% of ethanolic solution of AlCl3 and 3.0 mL (50 g/L) sodium acetate solutions were added. After 2.5 h of incubation at 20°C, the absorbance was read at 440 nm. The results were expressed as quercetin equivalent (mg/g) dried extract (QE/g dried ext).
Quantification of phenolic compounds by HPLC
High Performance Liquid Chromatography (HPLC) with UV detection is frequently used for the separation and detection of phenolic compounds in extracts. Samples were dissolved in pure water according to the ratio (0.3 g/10 mL) and centrifuged at 4706 rpm for 10 min. The supernatant was filtered through a cellulose acetate membrane filter (0.20 μm or 0.45 μm, Schleicher & Schuell) and used for analysis. The analysis was performed on an Agilent Technologies 1200 HPLC system fitted with a SUPELCOSIL LC-18 column (length 250 mm, diameter 4.6 mm, packaging size 5 mm). The column temperature was set at 20°C. The mobile phase consisted of a mixture of an aqueous solution of acetic acid (0.5%) by volume (“A”) and acetic nitrile (“B”). Elution was performed by using 100% of A for the first 2 min of the run, 40% of A and 60% of B from 2 to 60 min. The flow rate was set equal to 1 mL/min and the injection volume was 25 microlitres. Polyphenols were detected by a UV detector (280 nm). The retention times of the identified polyphenolic compounds of interest were measured by a single standard solution at a concentration of 100 mg/L.
Protective properties of the plant against oxidative damage
Preparation of liver homogenate
The liver was isolated from 3 normal albino Wistar rats. The organs were weighed and 10% (w/v) homogenate was prepared in phosphate buffer (0.1 M, pH 7.4 having 0.15 M KCl) using the homogenizer at 4°C [29]. The homogenate was centrifuged at 3000 rpm for 15 min and the clear cell-free supernatant obtained was used for the study.
Preparation of the pro-oxidative solution
The oxidant solution was prepared, directly before its utilization by adding a solution of ferric chloride 100 mM to H2O2 0.50% prepared in phosphate buffer (0.1 M, pH 7.4). This solution was used for the investigation of the protective assays on liver enzymes.
Total protein content
The total protein content of the mixture of liver was measured according to the protein kit supplier method (Human Kit-Hu102536, Boehringer Ingelheim, Germany). This result was used to express the activities of the different enzymes per gram of organs.
In vitro lipid peroxidation assay
Lipid peroxidation assay was performed by a formerly described protocol [50]. Phosphate buffer 0.58 mL (0.1 M; PH 7.4), 200 μL sample, 200 μL liver homogenate and 20 μL ferric chloride (100 mM) were combined to form mixture a which was placed in a shaking water bath for 1 h at 37°C. The reaction was terminated by adding 1 mL TCA (10%), TBA 1 mL (0.67%) to all the tubes which were placed in boiling water bath for 20 min. Then test tubes were shifted to crushed ice bath and were centrifuged at 3000 trs/rpm for 10 min. Absorbance of the supernatant was checked at 535 nm and was calculated as nM of MDA tissue using molar extinction coefficient of 1.56 × 105 /M.cm.
Determination of peroxidase activity
Peroxidase activity was determined by the peroxidase kit (CAS Number 7722-84-1, Sigma Aldrich) supplier with modifications. A solution containing the mixture of 1 mL of the oxidant solution (FeCl3, 100 mM) and extract or vitamin C (standard) for a final concentration of 100 μg/mL was incubated for 1 h in a water bath at 37°C. An aliquot of PBS (0.1 mL), hydrogen peroxide (50 μL), and pyrogallol solution (110 μL) were added to distilled water (625 μL) that was earlier dispensed into an Eppendorf tube. The plant extract (75 μL) from the mixture was thereafter added. For the blank, the control oxidant solution and the vitamin C as standard, the same reagents were used, except the extract which was replaced by distilled water (75 μL). The reaction was mixed and incubated for at least 10 min. The solution containing 100 mM, pH 6.0 PBS (40 μL) and 0.002% (v/v) diluted liver homogenate (40 μL) were added to the blank and test mixtures respectively. These were mixed, and the increase in absorbance at 420 nm was measured at every 10 s for 3 min using a spectrophotometer (BioMate 3S UV-Visible, Thermo Scientific™Manufacturer, Wohlen, Switzerland). One unit of peroxidase was defined as the change in absorbance/ seconds/mg of protein at 420 nm using molar extinction coefficient of 12 /M.cm.
Determination of catalase activity
Prior to the test, a solution containing a mixture of 1 mL of total volume of the oxidant solution and extract or vitamin C (standard) for a final concentration of 100 μg/mL was incubated for 1 h in a water bath at 37°C. The catalase activity of liver homogenate was assayed as previously described with modifications [51]. An aliquot of hydrogen peroxide (0.8 mL) was dispensed into an Eppendorf tube. Phosphate buffer (1.0 mL), extracted sample/Vitamin C/oxidant solution (75 μL) and (0.002% v/v) diluted homogenate (125 μL) were added. The reaction mixture (0.5 mL) was dispensed into 5% dichromate reagent (1.0 mL) and vigorously shaken. The mixture was heated in a Clifton water bath for 10 min, and allowed to cool. The absorbance at 570 nm was taken using spectrophotometer (BioMate 3S UV-Visible, Thermo Scientific™ Manufacturer, Wohlen, Switzerland). The absorbance obtained was extrapolated from the following standard curve y = 0.0028x + 0.0132. The catalase activity was thereafter expressed as Unit/min/mg of protein (UI/mg Prot.)
$$ CAT(unit)/ mg\; protein\Big)=\left( Abs/ \min \times 30000\; units\right)/\left(40 cm/M\times mgprotein\right)\times df $$
(5)
where df = dilution factor, Abs = absorbance.
Superoxide dismutase (SOD) activity
The measurement of total SOD activity was performed according to the Misra and Fridovich method with some slight modifications [52]. The principle of this method is based on the inhibition of epinephrine autoxidation. Distilled water (0.2 mL) and 2.5 mL sodium carbonate buffer 0.05 M, pH 10.2 were added to the 0.3 mL buffered epinephrine to initiate the reaction. The absorbance at 480 nm was read for 150 s at 30 s intervals against a blank made up of 2.5 mL buffer, 0.3 mL epinephrine and 0.2 mL distilled water. The following equation allowed the calculation of the SOD activity:
$$ SOD\left( unit/ mgprotein\right)= SO\left( unit s/ mL \min \right)/ protein\left( mg/ mL\right)\times df $$
(6)
where df = dilution factor.
The SOD activity was there after expressed as Unit/min/mg of protein (UI/mg Prot.).
Statistical analysis
The results were presented as mean ± SD of triplicate assays. Analyses of data was conducted using one-way ANOVA (Analysis of variance) followed by Kruskal wallis test and Dunnett’s multiple test (SPSS program version 18.0 for Windows, IBM Corporation, New York, NY, USA). The Log probit was used to determinate the IC50 using the software XLstat version 7 (Addinsoft, New York, NY, USA) were used to achieve the Pearson Correlation Analysis (PCA). The differences were considered as significant at P < 0.05.
