Influence of Growth Temperature on the Amounts of Tocopherols, Tocotrienols, and γ-Oryzanol in Brown Rice STEVEN J. BRITZ,* ,² P. V. V. PRASAD, ‡,§ ROBERT A. MOREAU, # L. HARTWELL ALLEN,JR., § DIANE F. KREMER, ² AND KENNETH J. BOOTE | Phytonutrients Laboratory, Beltsville Human Nutrition Research Center, Agricultural Research Service, U.S. Department of Agriculture, Beltsville Maryland 20705; Chemistry Research Unit, CMAVE, Agricultural Research Service, U.S. Department of Agriculture, Gainesville, Florida 32608; Eastern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, Wyndmoor, Pennsylvania 19038; and Agronomy Department, University of Florida, Gainesville, Florida 32611 Brown rice is a valuable source of lipid-soluble antioxidants including ferulated phytosterols (i.e., γ-oryzanol), tocopherols, and tocotrienols. To evaluate the impact of temperature on the accumulation of these compounds, seeds from six different rice lines grown to maturity in replicate greenhouses in Gainesville, FL, were analyzed. The lines represented Oryza sativa indica, O. sativa japonica, and Oryza glaberrima of different origins. Temperatures were maintained near ambient at one end of each greenhouse and at approximately 4.5 °C above ambient at the other end. γ-Oryzanols, tocopherols, and tocotrienols were extracted from whole seed (i.e., brown rice) and analyzed by HPLC. Tocotrienols and tocopherols varied widely between lines but changed only slightly with respect to temperature. In general, the proportions of R-tocotrienol and/or R-tocopherol increased at elevated temperature, whereas γ-tocopherol and γ-tocotrienol decreased. Six γ-oryzanol peaks, identified on the basis of absorbance maxima at 330 nm and HPLC-mass spectrometry, were quantified. The most abundant component was 24-methylenecycloartanyl ferulate, present at 40-62% of total. Its levels increased 35-57% at elevated temperature in five of six lines, accounting for most of the change in total γ-oryzanol. The results suggest that the physiological action of individual ferulated phytosterols should be investigated because their relative proportions in γ-oryzanol can change. KEYWORDS: Antioxidant; climate change; temperature; tocopherols; tocotrienols; oryzanol; Oryza sativa; Oryza glaberrima INTRODUCTION Rice bran constitutes about 10% of the dry matter of a seed and consists of the outer layers (pericarp, seed coat, nucellus, and aleurone) and the embryo or germ (1). Bran is about 15-20% oil, of which a relatively large proportion (ca. 4%) compared to other vegetable oils is unsaponifiable material, primarily free and esterified phytosterols as well as tocotrienols and tocopherols (collectively referred to tocols). The esterified sterols include 10 or more different compounds linked to ferulic acid (2) and are known collectively as γ-oryzanol. Similar to oat bran, rice bran and rice bran oil have significant hypocholesterolemic effects on humans and other species. Unlike oat bran, rice bran lipids and unsaponifiable components appear to be responsible (3-6). Isolated tocols, tocotrienols in particular (7), and γ-oryzanols (8) have both been implicated as active components in cholesterol reduction. Environment (i.e., planting location or planting year) as well as genetics has been reported to affect the contents and/or composition of tocols and/or γ-oryzanols in rice seed (9, 10). In addition to affecting product quality, such variation may have a significant effect on nutrition study outcomes if different sources of bran or oil are compared. Conversely, environmental manipulation in conjunction with genetic selection could be used to produce bran or oil varying in the content and composition of tocols and/or γ-oryzanols. The impact of environmental variables (e.g., temperature, drought, light, etc.) on rice phy- tochemical composition has not been determined. In soybean seeds, however, elevated temperatures (28 com- pared to 23 °C) or severe drought during seed development was shown to alter tocopherol metabolism, increasing the proportion of R-tocopherol (11). In other studies, seed development at 30 versus 15 °C resulted in 95% increases in total soybean phytosterols averaged across nine lines with a relative increase in campesterol compared to stigmasterol and -sitosterol (12). Large decreases in total tocopherols were also reported in * Corresponding author [e-mail steven.britz@ars.usda.gov; telephone (301) 504-6625; fax (301) 504-9456]. ² Food Components and Health Laboratory, U.S. Department of Agri- culture. ‡ Kansas State University. § Chemistry Research Unit, CMAVE, U.S. Department of Agriculture. # Eastern Regional Research Center, U.S. Department of Agriculture. | University of Florida. J. Agric. Food Chem. 2007, 55, 7559-7565 7559 10.1021/jf0637729 CCC: $37.00 © 2007 American Chemical Society Published on Web 08/29/2007