Enhanced Antioxidant Activity After Chlorination of Quercetin by Hypochlorous Acid Ralf Binsack, Brenda J. Boersma, Rakesh P. Patel, Marion Kirk, C. Roger White, Victor Darley-Usmar, Stephen Barnes, Fen Zhou, and Dale A. Parks Background: Several epidemiological studies indicate that moderate consumption of red wine decreases both the incidence and mortality associated with cardiovascular disease. Quercetin and rutin (quercetin- 3-rutinoside) are polyphenols present in relatively large concentrations in red wine and may play a role in this cardioprotective phenomenon. The precise mechanisms of cardioprotection remain unclear but may involve the action of these polyphenols as antioxidants, which attenuate the tissue injury that results from the production of proinflammatory oxidants such as hypochlorous acid (HOCl). Methods: To study the interaction of these polyphenols with proinflammatory oxidants, we mixed quercetin or rutin with HOCl (0 –150 M) and analyzed the reaction products by high-performance liquid chromatography, mass spectrometry, and nuclear magnetic resonance. Results: Stable mono- and dichlorinated derivates were detected for both quercetin and the glycoside derivative, rutin, which suggests that both the conjugated and unconjugated forms of quercetin reacted with HOCl similarly. Chlorination of quercetin occurred only at two sites, and the derivates (6-chloroquercetin, 6,8-dichloroquercetin) were more potent antioxidants toward oxidative modification of low-density li- poproteins and ABTS radical formation than the unmodified form. Conclusions: These data suggest that under certain pathological conditions in vivo (e.g., inflammation), flavonols may be converted to chlorinated derivates, which exhibit an enhanced antioxidant potential and thereby play a role in cardioprotection. Key Words: Quercetin, Rutin, Hypochlorous Acid, Chlorination, High-Performance Liquid Chroma- tography, Mass Spectrometry, Nuclear Magnetic Resonance. T HE FLAVONOLS QUERCETIN (3,3',4',5,7- pentahydroxyflavone) and rutin (quercetin-3-O--D- glucose-[1,6]-O--L-rhamnose; vitamin P) are present in relatively high levels in red wine, although they are not present in significant quantities in white wine (Goldberg et al., 1996; Soleas et al., 1997). Several epidemiological stud- ies indicate that the moderate consumption of red wine lowers the incidence and mortality associated with cardio- vascular disease (Fuhrman et al., 1995), due largely to a reduction in overall risk for coronary heart disease (Ca- margo et al., 1997; Doll, 1997; Klatsky et al., 1997; Thun et al., 1997). The 20% to 60% decrease in mortality has been attributed to the ability of moderate levels of alcoholic beverages to exhibit (a) antiatherogenic properties through an increase in high-density lipoprotein (HDL; McConnell et al., 1997), (b) antihemostatic properties by a decrease in platelet aggregation (Renaud and Ruf, 1996) and an in- crease in fibrinolytic activity (Aikens et al., 1998), and (c) antioxidant properties by minimizing oxidation of biomol- ecules such as lipids and proteins (Akkus et al., 1997; Croft, 1998; Keli et al., 1996; Svegliati-Baroni et al., 1999). It has been postulated that the red wine polyphenols quercetin and rutin also play an important role in this phenomenon (Nigdikar et al., 1998), although the precise mechanisms by which these flavonols result in cardioprotection remains unclear (Das et al., 1999; Miyagi et al., 1997). The antioxidant properties of flavonols are well recog- nized (Bravo, 1998; Croft, 1998), and it generally has been assumed that the cardioprotective potential of the flavonols is due, at least in part, to the scavenging of reactive oxygen species (Hertog et al., 1997; Serafini et al., 1998). In addi- tion, quercetin has been demonstrated to decrease super- oxide (O 2 -• ) and hydrogen peroxide (H 2 O 2 ) production by stimulated polymorphonuclear neutrophils (PMNs; Schnei- der et al., 1979), inhibit neutrophil degranulation (Black- burn et al., 1987; Pagonis et al., 1986), inhibit platelet aggregation stimulated by reactive oxygen species (Xie et al., 1996), reduce the phosphorylation of specific neutrophil From the Departments of Anesthesiology (RB, FZ, DAP), Pharmacology and Toxicology (BJB, SB), Pathology (RPP, VD-U), and Physiology and Biophysics (CRW), and The Comprehensive Cancer Center (MK), University of Alabama at Birmingham, Birmingham, Alabama. Received for publication August 14, 2000; accepted December 18, 2000. Supported in part by Grants AA11589 and AA12613 from NIH; the Alcohol Beverage Foundation and the Wine Institute; Instrumentation Grant S10RR06487 from NIH; and National Cancer Institute Core Research Grant P30 CA13148 for the University of Alabama at Birmingham Comprehensive Cancer Center. Reprint requests: Dale A. Parks, PhD, Department of Anesthesiology, 619 19th Street South, University of Alabama at Birmingham, Birmingham, AL 35233; Fax: 205-934-7437; E-mail: dale.parks@ccc.uab.edu Copyright © 2001 by the Research Society on Alcoholism. 0145-6008/01/2503-0434$03.00/0 ALCOHOLISM:CLINICAL AND EXPERIMENTAL RESEARCH Vol. 25, No. 3 March 2001 434 Alcohol Clin Exp Res, Vol 25, No 3, 2001: pp 434 –443