2.5. Determination of phenol content analysis of olive leaf extract Phenol content in olive leaf extract was determined using Folin–Ciocalteu reagent according to the method of Singelton, Orthofer,and Lamuela-Raventos (1999). Methanolic extracts were prepared as described in Section 2.4 and 100 ll of methanol was added to 100 ll of methanolic extract and reacted with 100 ll 1 N Folin reagent and 700 ll of freshly prepared 20% sodium carbonate in 1.5 ml micro-centrifuge tubes and the samples were vortexed briefly. The tubes were left to incubate in the dark for 20 min at room temperature and then centrifuged (Eppendorf 5417R, Hamburg, Germany) at 9464g for 3 min. The absorbance of the sample was read at 735 nm using aqueous gallic acid (10– 400 mg/ml) as a standard. Results were expressed as mg of gallic acid equivalent (GAE) per 100 g of dry weight of olive leaf extract.