Biological Activity of Acetylated Phenolic Compounds ELIZABETH FRAGOPOULOU, ² TZORTZIS NOMIKOS, ² HARALABOS C. KARANTONIS, ² CONSTANTINOS APOSTOLAKIS, ² EMMANUEL PLIAKIS, ² MARTINA SAMIOTAKI, § GEORGE PANAYOTOU, § AND SMARAGDI ANTONOPOULOU* ,² Department of Science of Nutrition-Dietetics, Harokopio University of Athens, 17671 Athens, Greece, and Protein Chemistry Laboratory B.S.R.C. “Alexander Fleming”, Athens, Greece In recent years an effort has been made to isolate and identify biologically active compounds that are included in the Mediterranean diet. The existence of naturally occurring acetylated phenolics, as well as studies with synthetic ones, provide evidence that acetyl groups could be correlated with their biological activity. Platelet activating factor (PAF) is implicated in atherosclerosis, whereas its inhibitors seem to play a protective role against cardiovascular disease. The aim of this study was to examine the biological activity of resveratrol and tyrosol and their acetylated derivatives as inhibitors of PAF-induced washed rabbit platelet aggregation. Acetylation of resveratrol and tyrosol was performed, and separation was achieved by HPLC. Acetylated derivatives were identified by negative mass spectrometry. The data showed that tyrosol and its monoacetylated derivatives act as PAF inhibitors, whereas diacetylated derivatives induce platelet aggregation. Resveratrol and its mono- and triacetylated derivatives exert similar inhibitory activity, whereas the diacetylated ones are more potent inhibitors. In conclusion, acetylated phenolics exert the same or even higher antithrombotic activity compared to the biological activity of the initial one. KEYWORDS: HPLC separation; acetylation; platelet activating factor; electrospray mass spectrometry; resveratrol; tyrosol INTRODUCTION Diet is the cornerstone of cardiovascular disease prevention. Although Mediterranean diets are associated with a low incidence of atherosclerotic disease, data about the specific dietary constituents involved and the mechanisms concerning cardioprotection are still sparse (1). Nutritional constituents that typically occur in small quantities in foods may be responsible for this effect (2). In recent years an effort has been made to isolate and identify a variety of biologically active compounds from foods included in the Mediterranean diet. Phenolic compounds present in all plants are considered to be biologically active constituents and have been studied extensively. Platelet activating factor (1-O-alkyl-2-acetyl-sn-glycero-3- phosphocholine, PAF) (3) is a potent inflammatory phospholipid mediator that is implicated in the initiation of the inflammation as well as in the process of atherogenesis (4). PAF and PAF- like lipids are degraded in plasma by the lipoprotein-bound enzyme, PAF acetyl hydrolase, leading to completely inactive compounds. In our previous studies we have isolated and characterized bioactive lipids from vegetable oils, olive oil, red/white wine, red/white must, fish, milk, and yogurt (5-10). These compounds had antithrombotic and anti-inflammatory actions because they inhibited or antagonized PAF (4). Even though these molecules have different origins, the biological activity has always been detected in the polar lipid fraction. Specifically, these molecules belong to phospholipids, glycolipids, phenolics, and phenolic glycosides. It should be noted that despite the different skeletons of these molecules, the majority of them had acetyl groups, which seems to be essential for their biological activity. Moreover, our data showed that the hydrolysis as well as the addition of acetyl groups in their structures led to differentiation of their biological action. The existence of acetylated phenolic compounds in natural sources is also established from other research groups. Acetyl- ated glycosides of flavonoids (quercetin, kaempferol, apigenin, luteolin) (11-14) and resveratrol (15, 16) have been found in plants, triacetylated resveratrol has been found in marine invertebrates (17), and acetylated tyrosol has been found in black olives (18) and olive oils (19). These data provide evidence about the existence of acetyl groups in naturally occurring compounds. Recently, attempts were made to correlate biological activity with the molecular structure. Structural modification of natural phenolics is expected to produce analogues that may be useful tools to study the structure-activity relationships. The synthesis of acetylated coumarins (20), acetylated O-glycosides (21), acetylated resveratrol (22) and its oligomers (23, 24), and * Address correspondence to this author at 70 El. Venizelou St., Athens 17671, Greece (telephone 0030-2109549230; fax 0030-2109577050; e-mail antonop@hua.gr). ² Harokopio University of Athens. § Protein Chemistry Laboratory B.S.R.C. “Alexander Fleming”. 80 J. Agric. Food Chem. 2007, 55, 80-89 10.1021/jf0627221 CCC: $37.00 © 2007 American Chemical Society Published on Web 01/03/2007