pubs.acs.org/JAFC Published on Web 09/15/2010 © 2010 American Chemical Society 10768 J. Agric. Food Chem. 2010, 58, 10768–10773 DOI:10.1021/jf102576j On the Inhibitor Effects of Bergamot Juice Flavonoids Binding to the 3-Hydroxy-3-methylglutaryl-CoA Reductase (HMGR) Enzyme MONICA LEOPOLDINI,NAIM MALAJ,MARIROSA TOSCANO,GIOVANNI SINDONA, AND NINO RUSSO* Dipartimento di Chimica, Universit a della Calabria, I-87036 Rende (CS), Italy Density functional theory was applied to study the binding mode of new flavonoids as possible inhibitors of the 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), an enzyme that catalyzes the four-electron reduction of HMGCoA to mevalonate, the committed step in the biosynthesis of sterols. The investigated flavonoid conjugates brutieridin and melitidin were recently quantified in the bergamot fruit extracts and identified to be structural analogues of statins, lipids concentration lowering drugs that inhibit HMGR. Computations allowed us to perform a detailed analysis of the geometrical and electronic features affecting the binding of these compounds, as well as that of the excellent simvastatin drug, to the active site of the enzyme and to give better insight into the inhibition process. KEYWORDS: HMGR; statin-like principles; enzyme inhibition; DFT INTRODUCTION Bergamot (Citrus bergamia Risso), a hybrid between orange and lemon plants, is a typical fruit of the southern provinces of Italy. It is mostly used for the extraction of its essential oil from the peel. Because of its unique fragrance and freshness, bergamot oil is widely used in the cosmetic and food industries ( 1 ). Further- more, due to its antiseptic and antibacterial properties, the essence is also used in the pharmaceutical industry ( 2 ). In the past few years, following the growing interest in anti- oxidant bioactive compounds and their dietary sources, bergamot juice, as well as its peel, has attracted some attention because of its remarkable content of flavonoids ( 3 -9 ). In particular, it has been identified and quantified ( 10 ) that together with the most abun- dant neoeriocitrin, naringin and neohesperidin, also C-glucosides (lucenin-2, vicenin-2, stellarin-2, lucenin-2-4 0 -methyl ether, scopar- in, and orientin 4 0 -methyl ether), flavone O-glycosides (rhoifolin 4 0 -O-glucoside, chrysoeriol 7-O-neohesperidoside-4 0 -O-glucoside, rhoifolin, chrysoeriol 7-O-neohesperidoside, and neodiosmin), and flavanone O-glycosides (eriocitrin) are present. Many epidemiological and biochemical studies ( 11 ) have dem- onstrated flavonoids to possess beneficial effects on human health because of their established antioxidant activity in scavenging harmful free radicals. Recently ( 12 ), a detailed analysis of ber- gamot fruit extractions, carried out by LC-MS, MS/MS, and NMR techniques, has led to the isolation of two new flavonoid conjugates, named brutieridin (hesperetin 7-(2 00 -R-rhamnosyl- 6 00 -(3 0000 -hydroxy-3 0000 -methylglutaryl)-β-glucoside)) and melitidin (naringenin 7-(2 00 -R-rhamnosyl-6 00 -(3 0000 -hydroxy-3 0000 -methylglu- taryl)-β-glucoside)) (1 and 2 in Scheme 1). They are present in bergamot fruit in concentration ranges of approximately 300-500 and 150-300 ppm, respectively, and are found either in the juice or in the albedo and flavedo layers of bergamot. Their structure resembles that of cholesterol inhibitor drugs statins ( 13 ). Like statins, the new active principles contain the hydroxyl mevalonate moiety. Mevalonate is a precursor of iso- prenoids, a class of compounds involved in several cellular func- tions such as cholesterol synthesis and growth control. Within cells, the concentration of mevalonate and therefore that of its metabolic products is tightly controlled through the activity of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), an enzyme that catalyzes the four-electron reduction of HMGCoA to mevalonate ( 14 ). Elevated serum-blood cholesterol levels are associated with a high risk of heart coronary artery diseases ( 15 ). Inhibition of HMGR enzyme is reported to significantly decrease cholesterol levels and to reduce the risks of stroke ( 16 ). Statins are potent HMGR inhibitors since they bind to the active site where also the natural substrate HMG binds, and are prescribed widely in the treatment of hypercholesterolemia. They are thought to bind competitively with the natural substrate in the active site of HMGR ( 17 , 18 ). However, adverse experiences have been asso- ciated with statins, both in monotherapy and in combination therapy with other agents ( 19 , 20 ). Both the wide variety of different types of flavonoids molecules and the fact that some of these compounds are present in good amounts (150-500 ppm) make possible the future applications of bergamot juice in health care, also considering the recommended statins dosage ranging from 5 mg to 80 mg, depending on the particular drug and cholesterol levels. The aim of this computational study is to investigate the interaction of the two new compounds brutieridin and melitidin with the active site of the human HMGR enzyme in order to have insights on their possible inhibitory effect. For the purpose of *To whom correspondence should be addressed. E-mail: nrusso@ unical.it.