Effect of Adlay (Coix lachryma-jobi L. var. ma-yuen Stapf) Testa and Its Phenolic Components on Cu 2+ -Treated Low-Density Lipoprotein (LDL) Oxidation and Lipopolysaccharide (LPS)-Induced Inflammation in RAW 264.7 Macrophages DIN-WEN HUANG, † YUEH-HSIUNG KUO,* ,‡,§ FANG-YI LIN, † YUN-LIAN LIN,* ,| AND WENCHANG CHIANG* ,† Graduate Institute of Food Science and Technology, Center for Food and Biomolecules, College of Bioresources and Agriculture, National Taiwan University, Taipei 106, Taiwan, Tsuzuki Institute for Traditional Medicine, College of Pharmacy, China Medical University, Taichung 404, Taiwan, Agricultural Biotechnology Research, Academia Sinica, Taipei 115, Taiwan, and National Research Institute of Chinese Medicine, Taipei 112, Taiwan, Republic of China The aims of this study were to investigate the effects of adlay testa (AT) on Cu 2+ -treated low-density lipoprotein (LDL) oxidation, 2,2′-diphenyl-1-picrylhydrazyl (DPPH)-scavenging capacity, and li- popolysaccharide (LPS)-induced inflammation in RAW 264.7 macrophages and determine its active components. The AT ethanolic extract (ATE) was partitioned into four fractions by various solvents as follows: n-hexane (ATE-Hex), ethyl acetate (ATE-Ea), n-butanol (ATE-Bu), and water (ATE-H 2 O). ATE-Ea and ATE-Bu were further fractionated into ATE-Ea-a-ATE-Ea-h and ATE-Bu-A-ATE-Bu- F, respectively, by column chromatography. Results showed that ATE-Ea, ATE-Bu, ATE-Ea-e, and ATE-Bu-C expressed antiradical, antioxidative, and anti-inflammatory activities with respect to the DPPH-scavenging capacity, LDL protection effect, and nitric oxide (NO) inhibitory activity. Inflammation was further modulated by ATE-Ea, ATE-Bu, ATE-Ea-e, and ATE-Bu-C through downregulating the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) proteins. The following components were found in ATE-Ea-e and ATE-Bu-C after purification and high-performance liquid chromatographic analysis: chlorogenic acid (CGA), vanillic acid (VA), caffeic acid (CA), p-coumaric acid (PCA), ferulic acid (FA), and 2-O--glucopyranosyl-7-methoxy-4( 2 H)-benzoxazin-3- one (GMBO). Results showed that CGA, CA, and FA were the major components responsible for the antioxidative and anti-inflammatory activities of ATE-Ea-e and ATE-Bu-C. Subsequently, each gram of ATE-Bu-C had 30.3 mg of CGA, 9.02 mg of CA, and 189 mg of GMBO, while each gram of ATE-Ea-e had 1.31 mg of VA, 3.89 mg of PCA, and 47.6 µg of FA. In conclusion, ATE has antioxidative and anti-inflammatory activities, and its effects are partially related to its phenolic components. Thus, ATE has the potential to be developed as a functional food targeting chronic diseases. KEYWORDS: Adlay; antioxidative; anti-inflammatory; LDL; DPPH; NO; iNOS; COX-2 INTRODUCTION Atherosclerosis is a chronic and complicated disease, leading to significant mortality in both the Western world and develop- ing countries. Many recent studies have noted that the increasing formation of reactive oxidative species (ROS) and inflammation play important roles in atherosclerosis. An increase in the low- density lipoprotein (LDL) content causes the early phase of atherosclerosis, according to the oxidative modification hypoth- esis; oxidation of LDL is the most likely cause of atherogenesis. The oxidation of LDL may be induced by various enzymes, free radicals, or other non-enzymatic mechanisms (1-3). Subsequently, oxidized lipids derived from LDL activate the nuclear factor (NF)-κB transcription factor, which was first identified by Sen and Baltimore (4), and this leads to the induction of the expression of many early genes, such as inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 * To whom correspondence should be addressed. Telephone: 886- 2-33661671. E-mail: yhkuo@ntu.edu.tw (Y.-H.K.); Telephone: 886- 2-28201999-6531. E-mail: yllin@nricm.edu.tw (Y.-L.L.); Telephone: 886-2-33664115. E-mail: chiang@ntu.edu.tw (W.C.). † National Taiwan University. ‡ China Medical University. § Academia Sinica. | National Research Institute of Chinese Medicine. J. Agric. Food Chem. 2009, 57, 2259–2266 2259 10.1021/jf803255p CCC: $40.75  2009 American Chemical Society Published on Web 02/25/2009