Inhibitory Effects of Safrole on Phagocytosis, Intracellular Reactive Oxygen Species, and the Activity of Myeloperoxidase Released by Human Polymorphonuclear Leukocytes

Abstract

Safrole, a component of Piper betle inflorescence, inhibits bactericidal activity and the release of superoxide anion (O(2)(-)) by polymorphonuclear leukocytes (PMNs). This in vitro study further investigated the effects of safrole on phagocytic activity, the intracellular production of reactive oxygen species (ROS), and the activity of the lysosomal enzyme myeloperoxidase (MPO), which is released by human PMNs. The possible effects of safrole on the phagocytic activity of PMNs against Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans) were determined using flow cytometry. PMNs were treated with various concentrations of safrole, which was followed by treatment with Hanks balanced salt solution with or without cytochalasin B and fMet-Leu-Phe (CB/fMLP). Intracellular ROS was determined using 2',7'-dichlorofluorescein diacetate and a fluorometer, whereas MPO activity was determined using a substrate assay. Safrole significantly inhibited the phagocytic activity of PMNs in a dose-dependent manner. Approximately 50% of the phagocytic activity of PMNs was affected when 10 mM safrole was used. Exposure of the PMNs to safrole (up to 5 mM) did not directly affect the intracellular levels of ROS and the extracellular activity of MPO. However, the ability of CB/fMLP to trigger production of intracellular ROS and the activity of MPO released by human PMNs was significantly suppressed by safrole. Safrole reduced the uptake of A. actinomycetemcomitans by human PMNs. Safrole also impaired the normal activation activity of PMNs. Alterations in the defensive properties of PMNs by safrole might promote bacterial colonization, and this could result in periodontal infection.