Biological Effects of Epicuticular Flavonoids from Primula denticulata on Human Leukemia Cells SERGEY V. TOKALOV, ² BARBARA KIND, ² ECKHARD WOLLENWEBER, § AND HERWIG O. GUTZEIT* ,² Institut fu ¨r Zoologie, Technische Universita ¨t Dresden, Mommsenstrasse 13, D-01062 Dresden, Germany, and Institut fu ¨r Botanik, Technische Universita ¨t Darmstadt, Schnittspahnstrasse 4, D-64287 Darmstadt, Germany The biological effects of epicuticular substances in farinose exudates accumulated on inflorescence shafts and calyces of Primula denticulata on human acute myeloid leukemia cells (HL-60) were analyzed. The crude material possessed little antioxidative capacity but strong cytostatic properties. Some of its known components (5-hydroxyflavone, 2′-hydroxyflavone, 5,2′-dihydroxyflavone, and 5,8- dihydroxyflavone) were further tested to identify the biologically active compounds. The effects of these flavones on cell cycle progression, mitochondrial membrane potential, and reactive oxygen species have been investigated by flow cytometry. The flavonol quercetin was included in the study as reference compound because of its known cytostatic properties and its activity as radical scavenger. Compared to quercetin the flavones induced little apoptosis (up to 40 μM), but despite their low toxicity, the Primula flavonoids possessed strong cytostatic properties even at low concentrations. The cell cycle distribution showed a characteristic time-dependent shift, giving evidence of a generally short- lived effect of the test compounds in the exposed cells. The antioxidative properties quantified according to two different methods correlated with the number of hydroxyl groups. Whereas quercetin strongly affected the mitochondrial membrane potential, none of the Primula flavones showed a comparable effect. KEYWORDS: Flavones; cell cycle; apoptosis; mitochondrial membrane potential; reactive oxygen species INTRODUCTION Primula is a plant genus with some 400 species, most of which are endogenous to the temperate zones. Some of them are popular garden plants because of their colorful early blossoms. The farinose leaf exudate of Primula species consists of a number of flavones with different biological activities (1). The induced activity of exudate compounds is illustrated dramatically by Primula dermatitis, which was first described by White more than 100 years ago (2). Allergic reactions were described mostly after contact with P. obconica. In most cases women and persons above the age of 35 were affected (3). The main sensitizer is the quinone primin, which is predominantly found in the distal cells of the microscopic glandular trichomes surrounding the calyx, but other allergens have been postulated (1, 4). Hausen et al. (4) isolated primetin (5,8-dihydroxyflavone) from P. mistassinica Michaux, the “birds eye primrose”, and demonstrated that this flavone had strong sensitizing properties. The substance has been detected in several other Primula species such as P. auricula, P. halleri, P. malacoides, P.marginata, and the species analyzed in this paper, P. denticulata (5). Many flavonoids have cytostatic properties (6) and may induce apoptosis (7). These activities have prompted numerous investigations to explore possible pharmaceutical applications. The cytostatic and apoptotic activities of the flavonoids have been attributed to their modulation of several biological processes. Flavonoids are potent inhibitors of processes involved in mitogen signaling or DNA synthesis, and this is thought to be the reason for the G 1 /S and/or G 2 /M arrests seen in some cell types following flavonoid exposure (8). Another sensitive and quite different indicator for cell toxicity is the induced reduction of the mitochondrial inner transmem- brane potential (∆Ψm). Recently, mitochondria have been shown to play a key role in apoptosis through the release of cytochrome c in response to many anticancer drugs and cellular stress (9). In many apoptotic pathways, the mitochondrial transmembrane potential collapses (see, for example, refs 10 and 11), indicating the opening of the so-called mitochondrial permeability transition pore. Even when a temporary reduction of the transmembrane potential does not result in apoptosis, the effect on the potential dependent proton pump presumably lowers adenosine 5′-triphosphate (ATP) production (12, 13). In this study we examined the effects of the flavonoids on the mitochondrial membrane potential using the fluorescent potential- sensitive dye JC-1. * Corresponding author (e-mail Herwig.Gutzeit@mailbox.tu-dresden.de; telephone 49 351 463 37536; fax 49 351 463 37093). ² Technische Universita ¨t Dresden. § Technische Universita ¨t Darmstadt. J. Agric. Food Chem. 2004, 52, 239-245 239 10.1021/jf0347160 CCC: $27.50 © 2004 American Chemical Society Published on Web 12/20/2003