Plant material
Barks of Syzygium guineense var. macrocarpum were harvested in the surrounding islands of the Sanaga River (Centre region- Cameroon) in November 2014 and identified at the National Herbarium of Cameroon under the reference number 49885 HNC.
Preparation of plant extracts
The harvested samples were cleaned, dried at room temperature and crushed. The powdered samples obtained were soaked separately in water, 95° ethanol, and the mixture water: ethanol (30:70;v/v) at pH = 3 for 48 h respectively. The mixtures were then filtered using Buchner funnel and Whatman N° 1 filter paper, concentrated under rotary evaporator. The aqueous and water:ethanol extracts were lyophilized while the ethanol extract was dried in an oven at 50 °C to obtain the crude extracts.
The resulting crude extracts were labelled as follows: SGFH2O: Syzygium guineense var macrocarpum aqueous extract (barks); SGFEtOH: Syzygium guineense var macrocarpum ethanolic extract (barks); SGF H2O/EtOH: Syzygium guineense var macrocarpum aqueous-ethanolic extract (barks). The crude extracts were stored at 4 °C until use. Before assaying each parameter, a stock solution of 1 mg/mL was prepared from which serial dilutions (0.025, 0.075, 0.150, 0.200 and 0.300 mg/mL) were prepared for the determination of the free radical scavenging activity. The phenolic metabolites content and antioxidant potential of different bark extracts were determined at 1 mg/mL.
Determination of free radical scavenging and antioxidant properties
Determination of free radical scavenging activity
Scavenging activity of DPPH radical
This assay measures the free radical scavenging potential of each crude extract. The method described by [35] was used. Briefly, 1000 μL of a 0.1 mM DPPH ethanolic solution was added to 3000 μL of each diluted extract or Vitamin C used as standard. After 30 min of incubation in the darkness at room temperature the absorbance was measured at 517 nm against a blank.
Scavenging effect of the ABTS+ radical
The radical scavenging capacity was measured by using ABTS+ solution radical cation. The assay was performed according to the method described by [36] with slight modifications. A stock solution of ABTS+ consisted of a 7.4 mM ABTS solution and 2.45 mM potassium persulfate solution in the ratio of 1:1. The mixture was allowed to react for 12 h at room temperature in the dark. A working solution was prepared by diluting 8 times the previous stock solution (20000 μL stock solution in 100000 μL volumetric flask, diluting it to the mark with ethanol) to get the absorbance of 0.7 ± 0.05 at 734 nm. After addition of 75 μL of extracts or vitamin C used as standard to 2000 μL of ABTS+ working solution, absorbance was measured at 734 nm after exactly 6 min.
The % inhibition for DPPH and ABTS assay was calculated according to the formula
$$ Scavening\kern0.5em effect\kern0.5em \left(\%\right)=100\times \left({A}_o-{A}_s\right)/{A}_o $$
Where Ao is the absorbance of the blank; As is the absorbance of the sample
Determination of antioxidant properties
Total antioxidant activity by Ferric Reducing Antioxidant Power assay (FRAP)
The FRAP assay was conducted following a previously described method [37] with slight modifications. The fresh FRAP reagent contained: acetate buffer (300 mM pH 3,6), 2,4, 6- Tri (2-pyridyl)-s-triazin (TPTZ) (10 mM) and FeCl3 · 6H2O (50 mM) in a 5:1:1 proportion respectively. FRAP reagent (2000 μL) was mixed to 75 μL of each tested extract and stored for 12 min. The activity of Vitamin C was used to plot the standard curve. The absorbance was read at 593 nm and results expressed as equivalent vitamin C/g of dried extract (mg eq Vit C/g DE).
Phosphomolybdenum antioxidant assay
The total antioxidant activity of extracts was evaluated by green phosphomolybdenum complex according to the method described by [38]. Phosphomolybenum reagent was prepared by mixing 0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate in 1:1:1 proportions. Phosphomolybdenum reagent (1000 μL) was introduced in test tubes. After addition of 10 μL of each extract sample, the mixture was homogeinized and the tubes incubated in a dry thermal bath at 95 °C for 90 min. Thereafter, tubes were cooled down and the absorbance of the mixture measured at 695 nm against a blank. The vitamin C was used as the standard and a calibration curve in the range of 0 – 0.3 mg/mL was prepared and BHT was used for the comparison. The reducing capacity of samples was expressed as mg of vitamin C equivalents/g of dried extract (mg vitC eq/g extract).
Determination of total phenol content
Total phenol content of the spice extract was determined using Folin-ciocalteu method [39]. This method is based on the reduction of phosphotungstate-phosphomolybdate reagent in alkaline medium. In different test tubes, 200 μL of 1 mg/ml of sample were introduced. Then, 800 μL of 10 fold diluted Folin reagent and 2000 μL of sodium carbonate solution (7.5 %) were added. After stirring, the mixture was kept away from light for 2 h and the absorbance was measured at 765 nm. The phenolic content was determined from a quercetin standard curve. A concentration range from 0 to 0.3 mg/mL of quercetin was prepared and allowed to determine the total polyphenol content expressed in mg equivalents of quercetin/g of extract (mg QE/g extract).
Determination of total flavonoid content
Total flavonoid content was determined using a well described method [40]. Briefly, 100 μL of extract were added to 300 μL of distilled water and 30 μL of NaNO2 (5 %). After 5 min of incubation at 25 °C, 30 μL of AlCl3 (10 %) were added. After further 5 min, the reaction mixture was treated with 200 μL of 1 mM NaOH and the reaction mixture diluted to 1000 μL with distilled water. Quercetin served to draw the standard calibration curve in the range of 0–0.3 mg/mL and the absorbance was measured at 510 nm. The results were expressed as mg quercetin equivalents/g of dried extract (mg QE/g extract).
Determination of total flavonol content
Total flavonols content in the plant extracts was determined according to the previously described technique [41] with slight modifications. In different test tubes, each extract (2000 μL) and standard solutions (2000 μL) were placed and then 2 % aluminum chloride (2000 μL), 50 g/L sodium acetate (3000 μL) were added and mixed well. The mixture was incubated at 20 °C for 2.5 h and absorbance was read at 440 nm. Total flavonols content was calculated as mg quercétine equivalent/g of extract using the equation based on the calibration curve and expressed as mg quercetin/g of dried extract (mgQE/g extract).
Determination of the polyphenolic content by HPLC
High Performance Liquid Chromatography (HPLC) with UV detection is frequently used to separate and characterize phenolic compounds present in extracts. The polyphenolic profile was determined according to a previously described method [28]. 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). Samples were dissolved in pure water to reach the concentration (300 mg/10 mL) and centrifuged at 4706 rpm for 10 min. The obtained supernatant was filtered through a cellulose acetate membrane filter (0.20 μm or 0.45 μm, Schleicher & Schuell). 25 μL of filtrate were injected into the HPLC system and eluted as described below.
The column temperature was set at 20 °C. The mobile phase consisted of a mixture of an aqueous solution of acetic acid at 0.5 % by volume (“A”) and acetic nitrile (“B”). Elution was performed by following this protocol: At start and for the first 2 min of the run, 100 % of A. From 2 to 60 min after the run start, a linear composition ramp was used, targeting 40 % of A and 60 % of B.
The flow rate was set to 1 mL/min. Polyphenols were detected by a UV detector (280 nm). Beforehand, the retention times of the identified polyphenolic compounds of interest available were measured by using of single standard solutions at a concentration of 100000 mg/mL. The quantification of identified compound was based on the area under peak determined at 280 nm and expressed relative to each corresponding phenolic standard.
Evaluation of organ protective effects of plant extracts
Preparation of different tissue homogenates
Normal albino wistar rats (10) were sacrified and the organs (liver, kidney, brain and heart) were isolated and weighed. Each homogenate was prepared by mixing 10 % (w/v) of each ground organ and phosphate buffer (pH 7, 0.1 M) followed by a centrifugation at 3000 rpm for 30 min. The study was approved by the Faculty of Medicine and Biomedical Sciences Ethical committee authorizing the use of animals.
Preparation of ferric-nitrilotriacetate solution
The oxidizing solution was prepared according to [42] Briefly,1.62 g and 7.64 g of FeCl3 and NTA were dissolved in 100000 μL of HCl 0,1 N to reach the concentrations of 200 mM and 400 mM respectively. The obtained solution was then mixed to a H2O2 200 mM 1:1 (v/v) The oxidant solution was prepared immediately before utilization.
Total protein content
The total protein content of the mixture of liver was measured according to the protein kit supplier methods (Human Kit-Hu102536, Boehringer, Ingelheim, Germany). This result was used to express the activities of the different enzymes per g of organs.
In vitro lipid peroxidation assay
The capacity of the spice extract to inhibit the lipid peroxidation was evaluated according to a previously implemented method [43]. In brief, 580 μL of phosphate buffer (0,1 M; pH 7,4), 200 μL of spice extract and 200 μL of each homogenate were successively introduced in different test tubes. Lipid peroxidation was then initiated by adding 20 μL of oxidizing solution (0.1 M HCl, FeCl3 200 mM, 400 mM NTA, 200 mM H2O2) in the mixture. The whole was thereafter placed in a water bath at 37 °C for 1 h. At the end of the incubation, 100 μL of this mixture was pipeted and placed in new tests tubes to which 1000 μL of MDA reagent (TCA (10 %) and 1 ml of TBA (0.67 %) were added to terminate the reaction. All the tubes were then heated again at 100 °C for 20 min and transferred to an ice bath to be cooled and centrifuged at 3000 rpm for 5 min. The optical density was measured at 535 nm and the concentration of MDA was calculated using the formula:
$$ \begin{array}{l}\mathrm{O}\mathrm{D} = \varepsilon \mathrm{C}\mathrm{l}\ \mathrm{and}\ \mathrm{expressed}\ \mathrm{in}\ \mathrm{n}\mathrm{M},\ \mathrm{where}\ \varepsilon\ \mathrm{molar}\ \mathrm{extinction}\ \mathrm{coefficient} = 1.56 \times 1{0}^5/\mathrm{M}/\mathrm{cm}\ \mathrm{and}\ \mathrm{l}=\mathrm{length}\ \mathrm{of}\\ {}\mathrm{the}\ \mathrm{tank}.\end{array} $$
Superoxide dismutase (SOD) activity assay
An indirect method of inhibiting autooxidation of epinephrine to its adrenochrome was used to assay SOD activity in plant-treated homogenates [44]. An aliquot consisting in (580 μL PBS, 200 μL of each extract or standard, 200 μL of liver, kidney, kidney, heart homogenate) and 20 μL of inducing solution was introduced in different test tubes and the obtained mixture was then incubated at 37 °C for 1 h to obtain the test solutions. The latter (test solutions) will be used to investigate the other enzymatic parameters as well as non enzymatic ones. To 20 μL of each test solution (Fe3+- NTA induced homogenates treated with plant extract or standard), 150 μL were added to 500 μL of carbonate buffer (pH 10.2; 0, 3 M; pKa 10.3), 250 μL of an EDTA solution (0.6 mM); The obtained mixture was then homogenized and 150 μL of an epinephrine solution (4.5 mm) were added to initiate the reaction. Four other tubes were run in the same conditions to serve as normal, negative and positive controls. The extract was replaced respectively by distilled water, oxidant, Vit C and quercetin.
The optical density was read after 30 min and 120 min at 480 nm. The following equation allowed the calculation of the SOD activity:
$$ SOD\left( unit/ mg\ protein\right)= SOD\left( unit s/ mL/ min\right)/ protein\left( mg/ mL\right)\times df $$
Where df = dilution factor
The SOD activity was thereafter expressed as Unit/min/mg of protein (UI/mg Prot.)
Catalase activity
The catalase activity of plant extracts on different homogenates was assessed according to a formerly described method [45] with some amendments. The above tests solutions (100 μL) were dispensed in test tubes containing 900 μL phosphate buffer (0.01 M, pH 7). After homogenization the reaction was started by the addition of 400 μL of a hydrogen peroxide solution (200 mM), and after 60 s, 2000 μL of an acetic acid-dichromate solution were added to stop the reaction. The mixture was boiled for 10 min and the absorbance was measured at 530 nm.
Glutathione peroxydase activity
In different test tubes, 580 μL of PBS (0.1 M; pH 7.4), 200 μL of each plant extract or vit C and quercetin used as standards, 200 μL of each homogenate (liver, heart, kidney and brain) and 20 μL oxidizing solution (HCl 0.1 M, FeCl3 200 mM, NTA 400 mM, H2O2 200 mM) were introduced. The normal control, negative and positive controls were run simultaneously in the same conditions except that, the oxidizing solution was replaced respectively by distilled water for the normal control, the plant extract by the distilled water for the negative control and vit C and quercetin for the positive controls. The mixtures were thereafter incubated at 37 °C for 1 h. Then, 100 μL of each of these mixtures were dispensed in new test tubes containing 900 μL of PBS (0,01 M; pH 7). An aliquot of PBS 0,01 M, pH 6; pH 7 (320 μL), hydrogen peroxide 0.05 % (160 μL), and pyrogallol solution 0.05 % (320 μL) were added to distilled water (210 μL). 100 μL from the above mixture was added thereafter. The reaction was mixed and incubated for at least 10 min and the increase in absorbance at 420 nm was measured after 20 and 140 s using a spectrophotometer.
Reduced glutathione assay
The previously described method of Ellman [46], was used to determine glutathione antioxidant capacity of plant extracts. An aliquot of PBS (580 μL), 200 μL of extract and 200 μL of each homogenate (liver, kidney, brain and heart) and 20 μL of inducing solution was introduced in different test tubes. The obtained mixture was then incubated at 37 °C for 1 h. The above test solutions (20 μL) and 3000 μL of Ellman reagent (phosphate buffer 0,1 M; pH 6,5; 2,2-dithio-5,5′-dibenzoïc acid) were introduced in new test tubes. Glutathione concentrations were expressed in micromoles/L and calculated using the following formula:
$$ \mathrm{O}\mathrm{D} = \varepsilon \mathrm{C}\mathrm{l}\ \mathrm{where}\ \varepsilon \mathrm{glutathione} = 13600\ \mathrm{and}\ \mathrm{l} = \mathrm{optical}\ \mathrm{path}. $$
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. XLstat version 7 (Addinsoft, New York, NY, USA) was used to achieve the Spearman rho Correlation Analysis as well as the principal component analysis (PCA). The differences were considered as significant at p < 0.05.
