Neuroprotective Effects of Quercetin 3-O-Methyl Ether, Quercetin and (±)-Dihydroquercetin in a Rat Model of Transient Focal Cerebral Ischemia

Abstract

Stroke is the third leading cause of death in most industrialized countries, and the most important source of chronic disability, among which cerebral ischemic stroke represents about 85% of all. Ischemic stroke occurs by thrombotic or embolic blockade of an artery to the brain resulting in a deficiency in blood flow and causing a brain infarction. During ischemic stroke, diminished blood flow initiates a series of events such as inducing excessive release of excitatory amino acids and subsequent several receptor activations leading to elevated calcium influx that may result in damage to brain cells. Oxidative stress is one of the primary factors that exacerbate damage in cerebral ischemia. Moreover, the reactive oxygen species can create a secondary source of extensive cell injury and death since re-supply of oxygen to the brain through the reperfusion is relevant during the ischemic phase. Therefore, antioxidants that can scavenge oxygen free radicals have the therapeutic potential for the treatment of neuronal injury following ischemia and reperfusion. Many natural antioxidants are reported to reduce reactive oxygen species and protect neuronal cells in animal models of cerebral ischemia. Previously, we reported protective effects of quercetin 3O-methyl ether (1) and its related compounds (2, 3) isolated from Opuntia ficus-indica var. saboten against oxidative neuronal injuries induced in primary cultured rat cortical cells (Fig. 1). Quercetin (2) was found to inhibit H2O2or xanthine (X)/xanthine oxidase (XO)-induced oxidative neuronal cell injury with an estimated IC50 value of 4-5 μg/ mL. (+)-Dihydroquercetin (3) inhibited oxidative neuronal injuries concentration-dependently, but it was 2-3 fold less potent than 2. On the other hand, quercetin 3-O-methyl ether (1) potently inhibited H2O2and X/XO-induced neuronal injuries with IC50 values of 0.6 and 0.7 μg/mL, respectively, indicating that 1 appeared to be the most potent neuroprotectant among three flavonoids isolated from this plant. Recently, antioxidative activities in cell-free systems of 1 have also been reported. However, neuroprotective effects of 1 in ischemic animal models have not yet been reported. The present study, therefore, examined neuroprotective effects of 1 in a transient focal cerebral ischemic rat model and compared with those of related flavonoids 2 and (±)-3. The middle cerebral artery occlusion (MCAO) model has usually been used for assessing neuroprotective effects since most ischemic strokes occur in the territory of the middle cerebral artery. Transient focal cerebral ischemia was induced by occlusion of the right middle cerebral artery for 2 h with a silicone-coated 4-0 nylon monofilament in male Sprague-Dawley rats. The antioxidative flavonoids dissolved in 0.9% saline vehicle were administered intraperitoneally at a dose of 10 mg/kg 30 min after onset of ischemia. Infarct size and % edema were measured 24 h after onset of ischemia using a 2,3,5-triphenyltetrazolium chloride (TTC) staining method. Neurological scoring was performed 30 min and 24 h after MCAO. The neurobehavioral tests consisted of scoring the degree of left forelimb flexion (0 to 3), the duration of left forelimb flexion (0 to 4) and symmetry of movement/forepaw outstretching (0 to 3). Rats were scored on a ranking scale of 0 to 10, which reflects the cumulative score of the individual tests with a score of 10 reflecting normal behavior. Representative TTC staining is shown in Figure 2 to illustrate neuroprotective effects of 1, 2 and (±)3. Area stained red with TTC was considered normal, while area not stained red with TTC was considered infarcted.