Plant materials and extraction
Leaves and fruits were harvested from Tounsi pomegranate trees in October 2021 from Mahdia region, Tunisia. Variety authenticity was confirmed by taxonomist Dr. Faten Zaouay from the Department of Horticulture, Higher Agronomic Institute, Chott-Meriem (University of Sousse, Tunisia) and a voucher specimen was deposited in our national collection maintained in duplicate at Gabes and Chott-Mariem (Sousse), with the code ‘TNl, TN2, TN3, TN5, TN5”.
Pomegranate extracts were prepared as described by our previous study [11]. Fruits were washed and hand-peeled. Arils were squeezed using a commercial blender (moulinex, France). The extract juice was centrifuged at 15000 rpm for 15 min. Then the supernatant was recuperated and lyophilized. Leaves, flowers and fruit peel were dried, powdered and extracted with methanol (MeOH) 50 g/250 ml in the dark for 48 hours. Each extract was filtered through Whatman No. 42 filter paper and evaporated to dryness using a rotary evaporator (Heidolph, Germany) under vacuum at 45 °C and stored at − 20 °C for further determination. Pomegranate seeds were dried and powdered. Oil was extracted by the methods of soxhlet. About 30 g seeds were extracted with 200 ml of hexane at room temperature for 6 h. The solvent was removed by evaporation at 40 °C and the oil was flushed with nitrogen stream and stored at − 20 °C in sealed tubes.
ABTS radical scavenging assay
The antioxidant capacity of pomegranate extracts by the ABTS (2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) assay was measured using a previous method [19]. Briefly, ABTS• + radical solution was produced by reacting the ABTS stock solution (5 mM) with potassium persulfate (K2S2O8) solution (2.7 mM). For the evaluation of antioxidant capacity, the ABTS• + solution was diluted with phosphate buffer (20 mM, pH 7.4) to obtain the absorbance of 0.700 ± 0.020 at 660 nm. Then, ABTS• + solution was mixed with pomegranate extracts prepared at different concentrations. After incubation, the absorbance was measured at 734 nm. Ascorbic acid was used as the positive control. The percentage of inhibition of ABTS• + radical was calculated with the following formula:
$$\textrm{Inhibition}\;\left(\%\right)=\left[\left(\textrm{A}\ \textrm{control}-\textrm{A}\ \textrm{sample}\right)/\textrm{Acontrol}\right]\ast 100$$
Acontrol refers to the solution containing pure MeOH instead of the sample, and Asample refers to the absorbance of pomegranate extract containing solutions. The effective concentration of sample necessary to decrease the absorbance ABTS• + by 50% (EC50) was determined.
Lipid peroxidation using ferric thiocyanate method
Inhibition of lipid peroxidation by pomegranate extracts was assayed according the previous procedure [20]. Linoleic acid (LA) was used as the lipid matrix and 2,2′-azobis (2-methylpropionamidine) dihydrochloride (AAPH) as the free radical initiator. Different concentrations of each pomegranate extract were prepared. Each concentration was mixed with 1.3% (w/v) methanolic LA and 0.2 M phosphate buffer (pH 7.0) and the peroxidation was initiated by the addition of AAPH solution (55.3 mM) in phosphate buffer. The control solution was prepared by adding pure MeOH instead of the sample. After incubation at 50 °C for 24 h in the darkness, the reaction mixture was dissolved in a 3:1 (v/v) H2O–MeOH solution. Then, a 10% aqueous solution of NH4SCN and 20 mM FeCl2 in 3.5% HCl were added. After 3 min of incubation at room temperature, the absorbance was measured at 546 nm against the corresponding blank. Ascorbic acid was used as the positive control. The results are expressed as the percentage of lipid peroxidation inhibition:
$$\%\textrm{Inhibition}=\left(\textrm{Acontrol}-\textrm{Asample}\right)\ast 100/\textrm{Acontrol}$$
Acontrol refers to the solution containing pure MeOH instead of the sample, and Asample refers to the absorbance of oil-containing solutions. The EC50 was determined.
Advanced glycation end-products inhibition assay
Inhibition of pentosidine-like AGEs formation and EC50 values were determined and calculated using a previously described method by Séro et al. 2013, with slight modifications [21]. Briefly, BSA (10 mg/mL) was incubated with D-ribose (0.5 M) together with the tested extract in 50 mM phosphate buffer at pH 7.4 (NaN3, 0.02%). Solutions were incubated in 96-well microtiter plates at 37 °C for 24 h in a closed system before AGE fluorescence measurement. Fluorescence resulting from the incubation, under the same BSA (10 mg/mL) and tested extract conditions, was subtracted for each measurement. Pentosidine-like (λexc 335 nm, λem 385 nm) AGEs fluorescence was measured using a microplate spectrofluorometer. The percentage of AGEs formation was calculated as follows for each extract concentration and the EC50 values were determined:
$${\displaystyle \begin{array}{l}\textrm{AGEs}\;\left(\%\right)=\left[\textrm{fluorescence}\ \textrm{intensity}\;\left(\textrm{sample}\right)-\textrm{fluorescence}\ \textrm{intensity}\;\left(\textrm{blank}\ \textrm{of}\ \textrm{sample}\right)\right]\\ {}{}^{\ast }100/\left[\textrm{fluorescence}\ \textrm{intensity}\ \left(\textrm{control}\right)-\textrm{fluorescence}\ \textrm{intensity}\left(\textrm{blank}\ \textrm{of}\ \textrm{control}\right)\right]\end{array}}$$
In vitro evaluation of anti-platelet aggregation activity
Fresh blood was obtained from healthy volunteers with negative history of consumption of drug, beverages or foods that may affect aggregation for at least 10 days and preferably should have fasted overnight because the presence of chylomicron may also disturb the aggregation patterns. The study was approved by the local ethics committee of the University Hospital Hedi Chaker of Sfax, Tunisia.
Venous blood was collected in a plastic tube containing trisodium citrate 109 mM. PRP was obtained by centrifuging at room temperature for 12 min at 200×g. PRP was removed carefully avoiding contamination with red cells or buffy coat, and stored at room temperature until tested. All the tests should be completed within 3 hours of preparing the PRP. The remaining blood was than centrifuged at 2000×g for 20 min to obtain platelet-poor plasma (PPP). We used a screening panel of aggregating agents: adenosine 5′-diphosphate (ADP, 20 μM), collagen (5 μg/mL) and arachidonic acid (2 mM).
PRP and PPP were used to set, respectively, 0 and 100% light transmission in the aggregometer. Platelet aggregation was monitored for at least 5 minutes after adding an agonist.
For pomegranate leaves (PL), flowers (PF), juice (PJ) and peel (PP) extracts, different concentrations were prepared previously for each extract dissolved in DMSO (at 0.05% final concentration). For PSO, different concentrations were dissolved in 70% Polyethylene glycol (PEG) which is a widely used solvent in an in vivo to dissolve water-insoluble compounds. Ten microliters of each extract were added to 260 μL of control PRP, and then the mixture was incubated for at least 5 minutes (until 30 min) at 37 °C before adding agonists. Then collagen (5 μg/mL), AA (2 mM) or ADP (20 μmol/L) was added and platelet shape change and aggregation were monitored for 5 min. DMSO (0.5% v/v) was used as negative control and aspirin was used as positive control.
The extent of platelet aggregation was calculated by the following formula:
$$\begin{array}{l}\text{Inhibition}\;\%=\left[1-\left(\text{D}/\text{S}\right)\right]\times100\end{array}$$
D = platelet aggregation in the presence of test compounds
S= platelet aggregation in the presence of solvent.
The platelet aggregation inhibitory activity was expressed as percent inhibition by comparison with that measured for the vehicle (DMSO or PEG) alone. Each sample was measured in triplicate and the data are presented as mean ± SD. The values of effective concentrations required for 50% inhibition of platelet aggregation (EC50), were obtained from at least three determinations.
Statistical analysis
Results were expressed as the mean of at least three independent measurements, unless standard deviations have been reported (means ± SD) and analyzed using SPSS ver. 21.0, professional edition. For antioxidant activities, Duncan’s test was used to estimate the significance among the main effects at the 5% probability level (P < 0.05).
