Materials and Methods Reagents. Crocetin was provided by Riken Vitamin Corpora- tion Limited (Tokyo, Japan). Hydrogen peroxide (H2O2) was purchased from Wako Pure Chem. Ind. (Osaka, Japan). The ESR spin trapping studies, using 5-(2,2-dimethyl-1,3-propoxycyclo- phosphoryl)-5-methyl-1-pyrroline-N-oxide (CYPMPO, Radical Research, Tokyo, Japan), indicated production of HO . Pento- barbital sodium was purchased from Kyoritsu Seiyaku Co. (Tokyo, Japan). MC-PROXYL was synthesized from 3-carboxy- 2,2,5,5-tetramethyl-pyrrolidine-1-oxyl (carboxy-PROXYL; Tokyo Kasei, Tokyo, Japan) by a method described previously.(25,37) All other reagents were analytical grades. In vitro ESR measurement. HO was generated by ultra- violet (UV, emission: 310–400 nm, 5 sec; 40 mW; SUPERCURE- 203S, RU-360, Radical Research, Tokyo, Japan) irradiation of H2O2 as described previously.(21,39,40) Crocetin was prepared in 10% alkaline buffer (50 mM Na2B4O7-50 mM Na2CO3, pH 10.0). Other solutions were prepared in ultra-pure water. ESR spin-trapping was conducted with an ROS-generating system containing CYPMPO.(41) ESR observations were performed with a JES-RE 3X, X-band spectrometer (JEOL, Tokyo, Japan) connected to a WIN-RAD ESR Data Analyzer (Radical Research, Tokyo, Japan) at the following instrument settings: microwave power, 8.00 mW; magnetic field, 335.6 ± 7.5 mT; field modulation width, 0.079 mT; receiver gain, 200; sweep time, 1 min; and time constant, 0.03 sec. All experiments were repeated a minimum of 3 times. For each experiment, the effects of the compounds were calculated and presented as the percentage of the mean control value (designated as 100%). Animal and ex vivo ESRCT imaging measurements. The procedures used in this study were in accordance with the guide- lines of the US National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publication NO. 85–23, revised 1985) and the protocols were approved by the Animal Care Committees (Yokosuka, Japan). Male SHRSPs (6-weeks old) were purchased from Japan SLC (Shizuoka, Japan). Animals were housed in a light-controlled room with a 12-h light/dark cycle and were allowed ad libitum access to food and water. Crocetin was suspended in 0.5% (w/v) carboxymethylcellulose-sodium (CMC- Na) solution at a concentration of 10 mg/ml (crocetin/CMC-Na). We have previously confirmed that crocetin arrived at the maximum blood concentrations 90 min after oral administration (data not shown). Crocetin (100 mg/kg) or 0.5% CMC-Na solution was administered orally 90 min prior to measurement by ex vivo ESR. ESR-computed tomography (CT) imaging of the isolated rat brain was performed as follows. The rats were anesthetized with 50 mg/kg (i.p.) pentobarbital and injected with 140 mmol/l MC-PROXYL solution (10 mg/kg) i.v. via the tail vein. The brain was isolated 30 sec after the treatment and subsequently analyzed using ex vivo L-band ESR imaging, as described previously.(26) Ex vivo ESR-CT imaging system constructed in our laboratory and JEOL ESR application laboratory software were used.(25,26,37,38) This system consists of a commercially available electromagnet (modified JES-RE 3X, JEOL, Tokyo, Japan), a pair of field scan coils, power supplies, a personal computer, and a 1-GHz micro- wave unit containing a 4-window loop-gap resonator (28 × ϕ43 mm, the measurement position centered on bregma). The system is provided with 4 different coil sets; 3 for the gradients (0.9 mT/cm, max) and 1 for rapid scanning. The gradient field was controlled by a current stabilizer linked with a personal computer (Dell Precision PWS 380). The ESR-CT images were constructed on the basis of Lauterburg’s method,(42) known as a 3D zeugmatography. We applied linear magnetic field gradients along the x-, y-, and z-axes produced by the magnetic field gradient coils. For the 2D imaging, 36 projections alternating between gradient and non-gradient were acquired in 55 s. Each projection required 1,024 points of F. Yoshino et al. acquisition data for imaging. The ESR absorption spectra were obtained by integrating the derivative spectrum with the recorded gradient. The mid-field hyperfine line in the spectrum was separated from the triplet signal of the nitroxyl radicals. Each signal data set was convoluted with Shepp’s filter function into the Fourier domain before performing the inverse Fourier trans- formation to the spatial domain. The 2D imaging pictures of 512 × 512 points were obtained from 18 projections per gradient step at 10° in the spatial domain. Instrument settings for ESR detection of MC-PROXYL were as follows: microwave power, 20 mW; magnetic field, 31.0–34.0 ± 1.0 mT; field modulation width, 0.1 mT; receiver gain, 63–125; time constant, 0.01 sec; field intensity, 0.7 mT/cm. Crocetin analysis in plasma and brain. Blood and brain was collected after crocetin administration orally 90 min later. After the collection of blood from common carotid artery, it was centrifuged at 1,500 g for 5 min at 4°C and plasma was separated. Brain was isolated after phosphate buffer saline perfused from the heart atrium. The samples were stored at −80°C. Plasma (100 μl) was mixed with 2.0 ml of methanol and centrifuged (3,000 rpm, 10 min). The supernatant was evaporated under nitrogen gas. We used the whole brain to analysis the crocetin distribution (control group: 1.44 ± 0.07 g wet weight, crocetin group; 1.67 ± 0.04 g wet weight) was homogenized in 2.0 ml of alkaline buffer and the homogenate was mixed with 6.0 ml of methanol/chloroform (1:1). The mixture was centrifuged (3,000 rpm, 10 min) and the supernatant was evaporated under nitrogen gas. The residue of plasma or brain was dissolved in 2.0 ml of alkaline buffer and loaded onto a solid-phase extraction cartridge (Oasis HLB Extraction Cartridge, Nihon Waters, Tokyo, Japan) pre-conditioned with methanol (2.0 ml) and alkaline buffer (2.0 ml). The cartridge was washed with water (2.0 ml) and hexane (2.0 ml). The analysis was eluted with methanol (2.0 ml) and the eluate was concentrated to dryness under nitrogen gas. The residue was reconstituted in 200 μl of methanol and filtered with a 0.45-μm Millipore filter for reversed-phase high performance liquid chromatography (HPLC) analysis. Crocetin was quantified by the HPLC method as described previously.(43) In recovery experiments, the recovery percentage of crocetin extracted from plasma and brain homo- genate was determined to be 99% and 92%, respectively. Statistical analysis. Results are expressed as mean ± SD. Student’s t test was used for comparisons between pairs of groups and Dunnet’s test was used for comparisons among 3 or more groups. Data were analyzed for statistical significance, and the significance level was set at p<0.05. Results Effects of crocetin on HO generation by H2O2 with UV irradiation. We investigated the effects of crocetin on HO , which had been generated from H2O2 by UV irradiation, and by ESR spin trapping with CYPMPO. In agreement with our previous report,(41) we observed that H2O2 generated by UV irradiation in the presence of CYPMPO led to the formation of a characteristic CYPMPO-OH spin adduct spectrum with hyperfine splitting giving rise to 14 resolved peaks (Fig. 2A). The generation of HO was not influenced by the 10% alkaline buffer (data not shown). As shown in Fig. 2B, CYPMPO-OH adduct formation was reduced in a dose-dependent manner by crocetin dissolved in 10% alkaline buffer (p<0.05). These data indicate that crocetin might be an effective HO scavenger. Effects of crocetin on SHRSPsinduced oxidative stress in the brain. MC-PROXYL is a suitable spin probe for the study of free radical reactions in the brain by in vivo and ex vivo ESR detection.(26,38) The effect of crocetin on SHRSPs-induced oxida- tive stress in the brain was investigated using MC-PROXYL and the resulting spectra were analyzed with the ESR-CT imaging system. Administration of crocetin to SHRSPs significantly J. Clin. Biochem. Nutr. | November 2011 | vol. 49 | no. 3 | 183 ©2011 JCBN