Original Article CtjJ01KOhcoCr980byreu.18iBon3ogrS20nt1Ni-on1a605,cal-4c0iJ0loe/08a1jAftce96pybCratfnilotci.n1rle1iiFc-ran0el1eBRioracdheiceaml diRsterusyea nrccdheNJuaptsraitniotnhe oxidative stress induced reactive oxygen species in the strokeprone spontaneously hypertensive rats (SHRSPs) brain Fumihiko Yoshino,1,† Ayaka Yoshida,1,† Naofumi Umigai,2 Koya Kubo2 and MasaichiChangil Lee 1,* 1Division of Pharmacology and ESR Laboratories, Department of Clinical Care Medicine, Kanagawa Dental College, 82 Inaokacho, Yokosuka, Kanagawa 2388580, Japan 2Riken Vitamin Co., Ltd., 51 Koji machi, Chiyoda, Tokyo 1020083, Japan (Re?ce?ived 14 January, 2011; Accepted 22 February, 2011; Published online 29 October, 2011) svCCtirrdoi2TCececa0hoetdte0iptsdvt?iynheireusiigssoaCehnrao,itgmond©ipainmset2atunorl0irnw0baas?ucloctcJriAeakoCsrntsBoit,srtaNieapbrnntruioodctipilodernerdcployirLsmotcirdcpiitueboecnudutst.nieodd,nouwifnhdgiaecanrhrydthepmeneiteraemdrfimirutumsito,sufpnatrnhoede- saffron, which has various effects in biological systems. In this study, we investigated the antioxidant effects of crocetin on reac tive oxygen species such as hydroxyl radical using in vitro Xband electron spin resonance and spin trapping. Crocetin significantly inhibited hydroxyl radical generation compared with the control. Moreover, we performed electron spin resonance computed tomography ex vivo with the Lband electron spin resonance imaging system and determined the electron spin resonance signal decay rate in the isolated brain of strokeprone spontane ously hypertensive rats, a highoxidative stress model. Crocetin significantly reduced oxidative stress in the isolated brain by acting as a scavenger of reactive oxygen species, especially hydroxyl radical, as demonstrated by in vitro and ex vivo electron spin resonance analysis. The distribution of crocetin was also determined in the plasma and the brain of strokeprone spontaneously hypertensive rats using highperformance liquid chromatography. After oral administration, crocetin was detected at high levels in the plasma and the brain. Our results suggest that crocetin may participate in the prevention of reactive oxygen speciesinduced disease due to a reduction of oxidative stress induced by reactive oxygen species in the brain. Key Words: crocetin, antioxidant, oxidative stress, brain, electron spin resonance (ESR) R RIenatcrtoivdeuocxtyiogenn species (ROS) such as the superoxide (O2 −) and/or hydroxyl radical (HO ) have been implicated in the pathogenesis of various types of brain dysfunction including ischemia-reperfusion injury,(1) Alzheimer’s disease,(2) aging,(3) and other neurodegenerative disease.(4) Among the organs that can be affected by ROS-induced diseases, the brain is particularly susceptible to the effects of aging and oxidative stress.(5) The brain protective properties of several carotenoids are well known.(6–9) It has recently been reported that antioxidant carotenoids such as β- carotene and lycopene reduce ischemia-reperfusion injury of the brain via their antioxidant properties.(10,11) Crocetin is a natural carotenoid compound found in the stigmas of saffron (Crocus sativus L.) and the fruits of Gardenia jasminoides Ellis. This yellow compound has been used as an im- portant spice and natural food colorant in various parts of the world.(12,13) In addition, saffron and gardenia fruits have been used as traditional medicine and crocetin is one of the major active compounds of these herbal medicines. Crocetin is an amphiphilic low-molecular weight carotenoid compound, as shown in Fig. 1. Extensive research on crocetin has indicated that it inhibits tumor promotion,(14) is hepatoprotective,(15) has neuroprotective doi: 10.3164/jcbn.1101 ©2011 JCBN Fig. 1. Chemical structure of crocetin. potential,(16) exerts anti-inflammatory effects,(17) and is beneficial in cardiac diseases.(18) In a recent clinical studies, crocetin showed positive effects on asthenopia(19) and attenuating effects on physical fatigue.(20) Antioxidant potential of crocetin may be contributing to these pharmacological actions. However, there are almost no reports on a direct ROS scavenging effect of crocetin. We previously reported on the use of an electron spin resonance (ESR)-based technique for the detection of free radical reactions in biological systems.(21–26) Nitroxyl radicals are very useful as spin probes for measuring ROS distribution, oxygen concentration, and redox metabolism by in vivo ESR in biological systems.(21–26) It has been reported that the nitroxyl radical, referred to as a ‘nitroxyl spin probe’, loses its ESR signal by rapidly reacting with HO (k>109 M−1 s−1),(27,28) O2 − (k = 104–105 M−1 s−1) in the presence of thiols or NAD(P)H,(28) and other radicals such as alkyl (k = 107– 109 M−1 s−1)(29) and lipid peroxyl radicals.(30) The signal decay rate of the nitroxyl spin probe provides evidence of ROS generation and changes in the redox status of biological systems.(31,32) The stroke-prone spontaneously hypertensive rat (SHRSP) is a genetic model of spontaneous hypertension, stroke, and endo- thelial dysfunction.(33,34) It has several characteristics of increased oxidative stress.(21,35–38) The blood brain barrier-permeable nitroxyl spin probe 3-methoxycarbonyl-2,2,5,5-tetramethylpyrrolidine-1- oxyl (MC-PROXYL) has been used for ESR assessment of oxidative stress in the rodent brain.(21,37,38) In the present study, we used the ESR technique to investigate the ROS scavenging effect of crocetin and the decay rate constant of MC-PROXYL in the isolated brain of the SHRSPs. In addition, we investigated the absorption and distribution of crocetin in the plasma and the brain following oral administration in SHRSPs. The results showed that oral administration of crocetin to SHRSPs was capable of reducing ROS-mediated oxidative stress in the brain due to a direct ROS-scavenging effect. *To whom correspondence should be addressed. Email: ieeman@kdcnet.ac.jp †These authors contributed equally to this work. J. Clin. Biochem. Nutr. | November 2011 | vol. 49 | no. 3 | 182–187