ANTIOXIDANT EFFECT OF FLAVONOIDS 963 Copyright © 2003 John Wiley & Sons, Ltd. Phytother. Res. 17, 963–966 (2003) Copyright © 2003 John Wiley & Sons, Ltd. Received 29 April 2002 Accepted 22 November 2002 PHYTOTHERAPY RESEARCH Phytother. Res. 17, 963–966 (2003) Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ptr.1289 SHORT COMMUNICATION Antioxidant Effect of Flavonoids on DCF Production in HL-60 Cells Satoshi Takamatsu, 1 * Ahmed M. Galal, 1 Samir A. Ross, 1 Daneel Ferreira, 1 Mahmoud A. ElSohly, 1 Abdel-Rahim S. Ibrahim 2 and Farouk S. El-Feraly 3 1 National Center for Natural Products Research and Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi, University, Mississippi 38677, USA 2 Department of Pharmacognosy, College of Pharmacy, Tanta University, Tanta, 8130 Egypt 3 Department of Pharmacognosy, College of Pharmacy, King Saud University, P.O.Box 2457, Riyadh 11451, Saudi Arabia A fluorometric microplate assay was performed for the detection of respiratory burst activity in a human leukaemia cell line HL-60 by assessing oxidation of 2′,7′-dichlorofluorescin diacetate (DCFH-DA). This method is based on the detection of DCFH oxidation due to the presence of hydrogen peroxide. In the present study, the antioxidant activity of a number of structurally related flavonoids of plant origin and some of their microbial transformation products (1–18) were evaluated. Copyright © 2003 John Wiley & Sons, Ltd. Keywords: respiratory burst activity; DCFH oxidation; antioxidants; flavonoids; HL-60 cell. * Correspondence to: Dr S. Takamatsu, National Center for Natural Pro- ducts Research and Research Institute of Pharmaceutical Sciences, School of Pharmacy, University of Mississippi, University, Mississippi 38677, USA. E-mail: stakamat@olemiss.edu. Contract/grant sponsor: United States Department of Agriculture; Contract/grant number: 58-6408-2-0009. technology is useful for the direct examination of the ability of natural products, e.g. polyphenols and flavonoids, to inhibit ROS in live human cells (Lee et al., 1998; Chung et al., 1999). The DCFH assay with flow cytometry was originally reported by Bass et al. for describing the intercellular detection of respiratory burst activity in phagocytic cells such as neutrophils and macrophages (Bass et al., 1983). A few researchers reported the application of the DCFH method for the evaluation of natural anti- oxidants (Lin et al., 2000; Rajbhandari et al., 2001). In a recent review, a method for visualizing the generation of oxidative species in whole animals was described (Tsuchiya et al., 1994). This paper reports the screening of a number of closely related flavonoids in our chem- ical library for antioxidant activity, the evaluation of this activity and the application of a fluorescent probe for high throughput screening (HTS). MATERIALS AND METHODS Chemicals. Carotene (α,β -mixture), vitamins E and C, and quercetin were purchased from Sigma. The isola- tion and preparation of the tested flavonoids were re- ported as follows: eriodictyol (1), 7-O-methylnaringenin (3), 7-O-methylapigenin (9), 7-O-methylluteolin (10), 7,3′-di-O-methylquercetin (11) and (2S,2″S)-7,7″-di-O- methyltetrahydroamentoflavone (12) (Ahmed et al., 2001); (2S)-5,3′,4′-trihydroxy-7-methoxyflavanone (4) and persicogenin (5) (Mossa et al., 1996); naringenin- 4′-sulphate (2), eriodictyol 4′-sulphate (6), homoerio- dictyol 7-sulphate (7) and naringin (8) (Ibrahim et al., in preparation); psiadiarabin (13), its glucoside (14), 5,3′-dihydroxy-7,2′,4′,5′-tetramethoxyflavone (15) and its glucoside (16) (Ibrahim et al., 1997); naringenin (17) and naringenin-7-sulphate (18) (Ibrahim, 2000). INTRODUCTION Reactive oxygen species (ROS) have been implicated in lipid peroxidation, inflammation, heart disease, catar- act, cognitive dysfunction, cancer and aging (Pietta, 2000). These disorders and diseases are a few of the long list that is continuing to grow as more scientists probe into the dark-side of molecular oxygen. This re- alization has stimulated a worldwide interest in endog- enous and exogenous antioxidants as defensive agents to combat the ROS generated (Colic and Pavelic, 2000). Flavonoids are well known as radical scavengers. The scavenging of ROS is essential for protecting against tissue damage of the vital organs. Thus the search for potent antioxidants, including antioxidant flavonoids, is necessary to diminish the cumulative effects of oxidative damage over the life span. Numerous in vitro solution-based chemical assay systems, such as the thiobarbituric acid (TBA) (Buege and Aust, 1978) colorimetric method, 1,1-diphenyl-2-picrylhydrazyl (DPPH) (Cuendet et al., 1997; Takao et al., 1994) and oxygen radical absorbing capacity (ORAC) (Wang et al., 1997) were reported for the evaluation of anti- oxidants. However, it is important to know how to feedback the results of in vitro assays into an evalua- tion of in vivo activity. Fluorescent technology has made it possible to evaluate antioxidants in live cells using specific probes such as 2′,7′-dichlorofluorescin diacetate (DCFH-DA). A cell-based method with fluorescent