Fig. 4. Effects of crocetin on oxidative stress in SHRSPs brain. SHRSPs were anesthetized with pentobarbital sodium (50 mg/kg, i.p.). The L band ESR was used to determine the signal decay of MCPROXYL in the isolated brain. Decay rate constants of MCPROXYL after the admin istration; control (0.5% CMCNa solution alone, open column), crocetin (suspended in 0.5% CMCNa solution administration, closed column). The results are expressed as mean ± SD (n = 6). * significant difference (p<0.05) versus the corresponding value in the control group. Table 1. Distribution of crocetin in plasma and brain Plasma Brain Group Control Crocetin Control Crocetin Concentration of crocetin nd 0.14 ± 0.05* nd 2.43 ± 0.40* Plasma and brain crocetin concentrations (mean ± SD) after the admin istration of 100 mg/kg crocetin suspended in 0.5% CMCNa solution (crocetin group) or 0.5% CMCNa solution alone (control group). Crocetin was not detected (nd) in plasma and brain of the control group. Results are expressed as mmol/l (in plasma) or nmol/g (in brain) (n = 6, respectively). * significant difference (p<0.0001) versus the corre sponding control value. and brain were both collected 90 min after the administration of crocetin. The crocetin concentrations in plasma and brain mea- sured by HPLC are shown in Table 1. These concentrations (about 0.14 mM in plasma and 2.43 nmol/g in brain) were significantly higher in the group that received crocetin as compared with the control group that did not receive crocetin. Discussion Various ROS may be generated by essential metabolic pro- cesses or by environmental stress such as UV exposure. Although the ROS plays important roles to cell signaling, it also has a potential to cause significant cellular damage. The participation of ROS in the pathogenesis of many diseases including brain dysfunction has been suggested.(1–4) Thus, in order to prevent ROS-induced disease, supplementation of antioxidants such as vitamin C, vitamin E, and carotenoids has been proposed.(44,45) Crocetin, a kind of carotenoid originally found in the dried stigma of saffron, has been used in the treatment of diversiform diseases for centuries.(46) It is well known that various carotenoids scavenge ROS such as HO and O2 −.(6–8) The carotenoids β- carotene and lutein have also been reported to inhibit ROS generation.(47) In recent years, it has been suggested that crocetin might be an effective antioxidant to counter oxidative stress in a hemi-parkinsonian rat model.(16) However, the antioxidant activity F. Yoshino et al. of crocetin appeared to involve the activation of the endogenous antioxidant enzymatic activities such as superoxide dismutase (SOD) and glutathione peroxidase (GPx),(16,48) rather than a direct ROS scavenging effect. There are few reports of the scavenging activity for ROS of the crocetine. Tseng et al.(49) reported the scavenging activity against O2 − using xanthine/xanthine oxidase system. ESR is an invaluable technique that provides a direct means of measuring the antioxidant effects. In this in vitro X-band ESR study, crocetin reduced the generation of HO from irradia- tion of H2O2 in a dose-dependent manner (Fig. 2). This study represents the first report of the scavenging effect of crocetin on HO . If we turn our attention to how our results may relate to the brain in vivo, it is critical to consider the relative concentration of the crocetin used in the present study. The concentration in rat brain of absorption of crocetin was about 2.43 nmol/g (Table 1), compared to the concentration of crocetin of 250 μM used in our in vitro experiments (Fig. 2). Indeed, it would be possibility that the scavenging effects of crocetin, much used in studies, may reach the brain. The redox potential of those unchanged crocetin and crocetin metabolites that reach the brain enables them to scavenge damaging radicals, but the endogenous brain antioxidants, especially ascorbate.(21) Regarding as the concentration of ROS generation on in vitro experiments, they would be much higher than in vivo situation. Brain tissue harvested from SHRSP is known to exhibit increased levels of oxidative stress.(21,35–38) Our present study has shown that the high concentration of crocetin treatment resulted in the recovery of normal levels of oxidative stress in the brain of SHRSP (Fig. 3 and 4). Other study revealed that crocetin would be helpful in preventing oxidative stress- induced neurologic disorder.(16,50) These results suggest the possibility that crocetin may show useful antioxidant activity for in vivo rodent model for human application. However, further studies will be required to examine the human application of crocetin upon oxidative stress-induced brain disease. The involvement of O2 − in ischemia-reperfusion injury, stroke, and atherosclerosis is well known.(51) It is possible to generate HO from O2 − via the Fenton reaction and/or Harber-Weiss reaction in biological systems.(52) These free radicals play an important role in brain damage after stroke. In addition to oxidizing macro- molecules, leading to cell injury, oxidants are also involved in cell death/survival signaling pathways and cause mitochondrial dysfunction.(1) Various antioxidants have been investigated for the treatment of stroke.(9,35) Crocetin, like other carotenoids, has the potential to be an effective treatment for diseases related to ROS, such as stroke, ischemia-reperfusion injury, and atherosclerosis. The SHRSP is a well-known model for atherosclerosis(53) and is useful for the study of oxidative stress caused by the generation of O2 − and HO in the brain. Our research group previously reported the utility of quantitative ESR analysis with MC-PROXYL for the assessment of redox status under conditions of oxidative stress in SHRSPs brain.(21,37,38) Spontaneous ROS generation has been demonstrated in association with ischemia-reperfusion injury accompanying atherosclerosis in SHRSPs brain.(36,53) In this study, we used ex vivo ESR-CT imaging to demonstrate the ability of crocetin (at a dose of 100 mg/kg) to reduce ROS generation and decrease the decay rate constant of MC-PROXYL in SHRSPs brain (Fig. 3 and 4). We assessed the metabolic fate and the bioavailability of crocetin in the plasma and the brain of SHRSPs and found that the compound was detected in both within 90 min after oral administration (Table 1). This result suggested that orally administrated crocetin may cross the blood-brain barrier and distribute to the brain. Taken together, these results indicate that crocetin attenuates oxidative stress in the isolated brain of SHRSPs. In conclusion, the present study demonstrated that crocetin exhibits antioxidant properties by scavenging ROS, and that it may reduce oxidative stress induced by ROS generation in the J. Clin. Biochem. Nutr. | November 2011 | vol. 49 | no. 3 | 185 ©2011 JCBN