G.J.B.B., VOL.4 (2) 2015: 145-152 ISSN 2278 – 9103 145 OPTIMIZATION OF EXTRACTION OF PHENOLIC AND ANTIOXIDANT CONTENTS FROM OLIVE LEAVES USING COMPOSITE CENTRAL DESIGN Nadia CHAMMEM a,1* , Ines SIFAOUI a,2 , Asma MEJRI 1 , Mourad Ben SLAMA 1 , Moktar HAMDI 1 , Manef ABDERABBA 2 1 Laboratoire d’Ecologie et de Technologie Microbienne, INSAT, University of Carthage 2 Laboratoire Matériaux-Molécules et Applications, IPEST, University of Carthage. a These authors contributed equally to this work *Corresponding author’s e-mail: ines.sifaoui@hotmail.com ABSTRACT Recently, the interest towards phenol compounds has progressed. Olive leaves have been used from the past as cure against several pathologies. Still, the extraction of the bioactive molecules from this plant material should be investigated. In this study, a Response surface methodology (RSM) was applied to predict the optimum conditions for extraction of phenolic and antioxidant compounds from olive leaves. Two independent variables, solvent composition and temperature, were investigated. The experimental data were fitted to a second-order polynomial equation. Optimal extraction conditions were as follows: solvent concentration of 80% for ethanol and 75% for methanol, extraction temperature of 40°C for ethanolic extract and 50°C for methanolic extract. Under these conditions, the experimental yield of extracted polyphenols was 51 mg EAG g -1 dp and 56 mg EAG g -1 dp as for the experimental antioxidant activity was 90% and 92% respectively for ethanolic and methanolic extracts. KEY WORDS: RSM, Olive leaves, extraction, phenolic compounds, antioxidant activity. 1. INTRODUCTION Recently, many human illnesses such as cancer, heart and cerebrovascular diseases are correlated to Reactive Oxygen Species (ROS). In fact, those molecules are generated from auto-oxidation and thermal oxidation of different metabolism like the lipids (Frang et al., 2002; Briante et al., 2003; Huang et al., 2005). A wide array of enzymatic and non-enzymatic endogenous antioxidant defense systems has been evolved to compensate for the generation of ROS (Sies, 1997). Recently, Common natural antioxidants include tocopherols, carotenoids, flavonoids, and polyphenols - compounds have received considerable attention especially for they function as chemo preventive agents against oxidative damage (Carrasco-Pancorbo et al., 2005; Perez-Bonilla et al., 2006). Among the plants rich in antioxidant compounds the olive tree (Olea europeaea, Oleaceae) has historically provided huge economic and dietary benefits to the Mediterranean basin (Japòn-Lujàn et al., 2006). In fact, olive oil as well as leaves have been used for medical purposes, and were introduced recently into the Pharmacopoea PhEur 5. Olive Leaf Extract (OLE) has also been used by native people of these areas in folk medicine to treat fever and other diseases such as malaria (Gucci et al., 1997; Fernández-Escobar et al., 1997; Ciafardini and Zullo; 2002). Recently, several works have confirmed the pharmacology effects of (OLE). Among those reports some have shown that (OLE) has the capacity to lower blood pressure in animals (Samuelsson, 1951) to increase blood flow in the coronary arteries (Zarzuelo et al., 1991) to relieve arrhythmia, and to prevent intestinal muscle spasms (Benavente-Garcia et al., 2000). Recent studies have focused on the olive leaves contents and their extraction as high-added value compounds (Da Silva et al., 2006; Japòn-Lujàn et al., 2006; Ranalli et al., 2006). Solvent extraction is a favorable process since heat sensitive materials can be recovered at low temperatures. In order to optimize the recovery of the bioactive compounds, the parameters affecting the extraction process should be investigated. This study aims to investigate the efficiency of extraction of polyphenols and antioxidants from olive leaves. Central Composite Design (CCD) was used to investigate the effects of two independent variables - namely solvent composition and temperature (°C) for maceration - on phenols and antioxidant content. 2. MATERIALS & METHODS 2.1. Samples The Swebea Elgia variety was used to determine the experimental conditions for the phenolic and antioxidant extraction method. 2.2. Extraction protocol The olive leaves collected during the maturing fruit season of 2009/2010 were ground to a fine powder using a mill. 1.2.1. Maceration Each shell dry powder sample (0.5 g) with 18% of humidity was macerated with 5mL of extraction solvents in a capped glass tube on an agitating plate (IKA ®KS 130 basic, Chine) at a constant speed (0.03g) for 24 hours. The glass tube was wrapped in aluminum foil to prevent light degradation during extraction. Extraction solvents used were methanol, ethanol and distilled water. Afterwards, a rotary vacuum evaporator (Heidolph, Germany) set at