Assessment of antioxidant, anti-inflammatory, anti-cholinesterase and cytotoxic activities of pomegranate (Punica granatum) leaves Jalila Bekir a,b , Mohamed Mars b , Jean Pierre Souchard a , Jalloul Bouajila a,⇑ a Université de Toulouse, Laboratoire des Interactions Moléculaires et Réactivité Chimique et Photochimique UMR CNRS 5623, Université Paul-Sabatier, 118 route de Narbonne, F-31062 Toulouse, France b Unité de recherche: Biodiversité et Valorisation des Bioressources en Zones Arides. Faculté des sciences de Gabès. Cité Erriadh Zrig, 6029 Gabès, Tunisia article info Article history: Received 6 December 2012 Accepted 24 January 2013 Available online 1 February 2013 Keywords: Punica granatum Anti-inflammatory Antioxidant Cytotoxic Cholinesterase Extraction abstract This study evaluated antioxidant, anti-inflammatory, anti-cholinesterase and cytotoxic activities of extracts with different polarities (hexane, dichloromethane, ethyl acetate, ethanol and methanol) obtained from Punica granatum leaves. Total phenolics (8.8–127.3 mg gallic acid equivalent/g dry weight), flavonoids (1.2–76.9 mg quercetin equivalent/g dry weight), tannins (63.7–260.8 mg catechin equivalent/kg dry weight) and anthocyanins (0.41–3.73 mg cyanidin-3-glucoside equivalent/g dry weight) of different extracts were evaluated. The methanolic extract presented a good IC50 by DPPH and ABTS assays (5.62 and 1.31 mg/l respectively). The strongest 5-lipoxygenase (5-LOX), acetylcholines- terase (AChE) and butyrylcholinesterase (BuChE) inhibition activities were obtained for the ethanol extract (IC50 values of 6.20, 14.83 and 2.65 mg/l, respectively) and the best cytotoxic activity against MCF-7 cells was obtained for the methanol extract (IC50 = 31 mg/l). These important biological activities showed that P. granatum leaves could be a potential source of the active molecules intended for applica- tions in pharmaceutical industry, but only after additional in vivo experiments. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction Oxidative stress is defined as the imbalance between pro-oxi- dants and antioxidants, in favour of the former. Numerous studies have shown that the oxidative stress contributes significantly to the development and the progression of wide variety of diseases such as diabetes, cancer and neurodegenerative, cardiovascular, and pulmonary diseases (Toullec et al., 2010). Christen (2000) re- ported that free radicals are likely an important factor in the path- ogenesis of Alzheimer disease (AD). The brains of AD patients present several signs of free radical attacks such lipid peroxidation, protein oxidation, damage to mitochondrial and nuclear DNA. Also, the crucial role of superoxide production in the pathogenesis of diabetes was pointed out (Brownlee, 2001). New clinical and epi- demiological data consider the oxidative stress as an important determinant in the development of chronic inflammation and can- cer through activation of variety of transcriptional factors (Reuter et al., 2010; Toullec et al., 2010). Epidemiological studies reported also that antioxidants play a key role in protecting against oxida- tive damage. Thus, antioxidants presents in fruit and vegetable were well recommended to reduce cardiovascular and neurode- generative risks (McCall et al., 2011). These positive health effects were attributed to a variety of compounds such as phenolics and terpenoids. In recent years, considerable attention has been direc- ted to study these active compounds from medicinal plants for var- ious pharmacological purposes (Dorman et al., 2004). The different parts of pomegranate (Punica granatum L.) have been known as a reservoir of bioactive compounds with potential biological activities. Pomegranate, especially the leaves of pome- granate, decreased the dyslipidemia of obesity and cardiovascular risk factors (Lei et al., 2007). Anti-parasitic, anti-microbial and antioxidant activities of pomegranate leaves extracts were re- ported (Egharevba et al., 2010; El-Shennawy et al., 2010 and Wang et al., 2013). Several papers were reported on the ability of pomegranate leaves extracts to fight obesity (Al-Muammar and Fozia Khan, 2012), cancer and other human diseases (Lansky and Newman, 2007). Phenolics in pomegranate leaves are thought to contribute in their health benefits (Lan et al., 2009). To our knowledge, no study in literature for anti-inflammatory, anti-cholinesterase inhibitory and cytotoxic activities of P. grana- tum leaves. The objectives of this study were to determine the quantifica- tion of the main classes of polyphenolic compounds of pomegran- ate leaves extracts prepared with different polarity solvents and to compare antioxidant, anti-cholinesterase, anti-inflammatory and cytotoxic activities of different extracts. 0278-6915/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.fct.2013.01.036 ⇑ Corresponding author. Tel.: +33 562256825; fax: +33 562256826. E-mail address: jalloul.bouajila@univ-tlse3.fr (J. Bouajila). Food and Chemical Toxicology 55 (2013) 470–475 Contents lists available at SciVerse ScienceDirect Food and Chemical Toxicology journal homepage: www.elsevier.com/locate/foodchemtox