Antimutagenicity of Supercritical CO 2 Extracts of Terminalia catappa Leaves and Cytotoxicity of the Extracts to Human Hepatoma Cells TING-FU KO, † YIH-MING WENG, † SHWU-BIN LIN, § AND ROBIN Y.-Y. CHIOU* ,# Graduate Institute of Food Science and Graduate Institute of Biotechnology, National Chiayi University, Chiayi, Taiwan, and Graduate Institute of Medical Technology, Department of Laboratory Medicine, National Taiwan University Hospital, College of Medicine, National Taiwan University, Taipei, Taiwan Natural antimutagens may prevent cancer and are therefore of great interest to oncologists and the public at large. Phytochemicals are potent antimutagen candidates. When the Ames test was applied to examine the antimutagenic potency of supercritical carbon dioxide (SC-CO 2 ) extracts of Terminalia catappa leaves at a dose of 0.5 mg/plate, toxicity and mutagenicity were not detected. The antimutagenic activity of SC-CO 2 extracts increased with decreases of temperature (60, 50, and 40 °C) and pressure (4000, 3000, and 2000 psi) used for extraction. The most potent antimutagenicity was observed in extracts obtained at 40 °C and 2000 psi. At a dose of 0.5 mg of extract/plate, approximately 80% of the mutagenicity of benzo[a]pyrene (B[a]P, with S-9) and 46% of the mutagenicity of N-methyl-N ′-nitroguanidine (MNNG, without S-9) were inhibited. Media supplemented with SC-CO 2 extracts at a range of 0-500 μg/mL were used to cultivate human hepatoma (Huh 7) and normal liver (Chang liver) cells. The viability of the cells was assayed by measuring cellular acid phosphatase activity. A dose-dependent growth inhibition of both types of cells was observed. The SC-CO 2 extracts were more cytotoxic to Huh 7 cells than to Chang liver cells. The observation that SC-CO 2 extracts of T. catappa leaves did not induce mutagenicity at the doses tested while exhibiting potent antimutagenicity and were more cytotoxic to human hepatoma cells than to normal liver cells is of merit and warrants further investigation. KEYWORDS: Antimutagenicity; Terminalia catappa; supercritical fluid extraction (SFE); Ames test; cytotoxicity; human hepatoma; Chang liver cells INTRODUCTION Known and unknown mutagens and antimutagens present in the environment may affect the incidence of human cancer. In addition to contact with skin, ingestion is the most frequent channel of human exposure. Interactions among bioactive compounds are complicated and ubiquitous, making the detec- tion and identification of natural mutagens and antimutagens important (1, 2). When people are aware of sources of natural antimutagens, they are more likely to make selections of food or drink containing substantial amounts of active compounds, thereby enhancing their health status. Intake of sufficient amounts of antimutagens and/or anticarcinogens is believed to confer a preventive effect on the initiation and development of human cancers (3). Phytochemicals are potent sources of antimutagens. Terminalia catappa, belonging to the family Combretaceae, is a tree that commonly grows in tropical and subtropical regions. The fallen leaves, after drying and shredding, have been used as a folk medicine (4) and also as a drink after infusion with hot water. Water extracts of T. catappa leaves have been reported to effectively suppress CCl 4 -induced hepatotoxicity of male Wistar albino rats and bleomycin-induced genotoxicity of Chinese hamster ovary cells (4, 5). Punicalin and punicalagin, both anti-AIDS compounds, have been identified in the water extracts of T. catappa leaves (6, 7). Tannin and flavonoid glycosides in T. catappa leaves, as free radical scavengers, have been reported to inhibit Cu 2+ -induced low-density lipoprotein oxidation (8-10). Extraction and identification of squalene from T. catappa leaves by supercritical carbon dioxide (SC-CO 2 ) have been achieved in our laboratory (11). In addition to the potent antioxidative characteristics of SC-CO 2 extracts, further inves- tigation of the antimutagenicity of extracts was of interest. Supercritical fluid extraction (SFE) has been demonstrated to effectively extract natural and neutraceutical compounds (12). SFE is a powerful tool in the differential and efficient extraction of the target compounds from solid matrices at a fairly low * Corresponding author (telephone 8865-2717613; fax 8865-2775524; e-mail rychiou@mail.ncyu.edu.tw). † Graduate Institute of Food Science, National Chiayi University. § National Taiwan University Hospital. # Graduate Institute of Biotechnology, National Chiayi University. 3564 J. Agric. Food Chem. 2003, 51, 3564-3567 10.1021/jf034102v CCC: $25.00 © 2003 American Chemical Society Published on Web 05/10/2003