Inhibitory Effects of Safrole on Phagocytosis, Intracellular Reactive Oxygen Species, and the Activity of Myeloperoxidase Released by Human Polymorphonuclear Leukocytes Lien-Yu Chang,* † Jung-Chen Lin, ‡ Ching-Wen Chang, § Weng-Hang Ho, ‡ Yen-Ting Chen, ‡ Ji-Lung Peng, ‡ and Shan-Ling Hung †‡ Background: Safrole, a component of Piper betle inflores- cence, inhibits bactericidal activity and the release of superox- ide 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 oxy- gen species (ROS), and the activity of the lysosomal enzyme myeloperoxidase (MPO), which is released by human PMNs. Methods: The possible effects of safrole on the phagocytic activity of PMNs against Aggregatibacter actinomycetemcomi- tans (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 29,79-dichlorofluorescein diacetate and a fluorometer, whereas MPO activity was determined using a substrate assay. Results: 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 extracel- lular 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. Conclusions: Safrole reduced the uptake of A. actinomyce- temcomitans by human PMNs. Safrole also impaired the normal activation activity of PMNs. Alterations in the defensive proper- ties of PMNs by safrole might promote bacterial colonization, and this could result in periodontal infection. J Periodontol 2009;80:1330-1337. KEY WORDS Myeloperoxidase; phagocytosis; polymorphonuclear leukocytes; reactive oxygen species; safrole. P eriodontal disease results from the interaction between the host-de- fense mechanisms and the micro- organisms in the dental plaque biofilm. 1 Polymorphonuclear leukocytes (PMNs), the most abundant circulating leuko- cytes, represent the first line of these host-defense mechanisms. The main functions of PMNs include adherence, chemotaxis, phagocytosis, and bacteri- cidal activity. 2 Patients with altered numbers or functionality of circulating PMNs tend to have severe periodontal disease. 3 Approximately 1 million PMNs enter the oral cavity via the gingival crevicular fluid every minute. 4 The PMNs within the gingival crevice are able to protect the gingiva against mi- crobial invasion through mechanisms such as secreting hydrolytic enzymes and producing oxygen radicals that are directed toward microbial killing. 2,5 The production of reactive oxygen species (ROS), such as superoxide, hydrogen peroxide, and hydroxyl radical, by a PMN respiratory burst is an important pathway associated with oxidative kill- ing. 3,6 Exocytosis of granules and se- cretory vesicles (degranulation) also play pivotal roles in PMN activity from early activation to the destruction of * Department of Dentistry, National Yang-Ming University, Taipei, Taiwan. † Department of Education and Research, Taipei City Hospital, Taipei, Taiwan. ‡ Institute of Oral Biology, National Yang-Ming University. § Department of Dentistry, Taipei City Hospital. doi: 10.1902/jop.2009.080202 Volume 80 • Number 8 1330