Wet tomato pomace powder consisting of tomato skins, pulp and crushed seeds that remain after the processing of tomato for juice was dried with oven at 50 degree Celsius for 2 hours. Then, it was grinded as powder and determined total phenolic compound and used as marker for quality control. The dried tomato pomace powder at the percent yield of 12.75 was kept in airtight container at room temperature until use. The administration of dried tomato pomace powder was performed using propylene glycol as vehicle.

The total phenolic content of extracts was determined using to the Folin-Ciocalteu method [9]. The extracts were oxidized with Folin-Ciocalteu reagent, and the reaction was neutralized with sodium carbonate. The absorbance of the resulting blue color was measured at 760 nm after 60 min. Using gallic acid (GA) as standard total phenolic content (standard curve was prepared using concentrations of 2.5 - 50 mg/ml) was expressed as mg GA equivalent/mg of extract. Data reported of three replications.

Male Wistar rats weighing 300 - 350 gm were obtained from National Laboratory Animal Center, Salaya, Nakorn Pathom, and were housed in group of 5 per cage in standard metal cages at 22˚C ± 2˚C on 12:12 h light-dark cycle. All animals were given access to food and water ad libitum. The experiments were performed to minimize animal suffering in accordance with the internationally accepted principles for laboratory use and care of European Community (EEC directive of 1986; 86/609/EEC). The experimental protocols were approved by the Institutional Animal Care and Use Committee.

In order to induce focal cerebral ischemia, all rats in this study were occluded the right middle cerebral artery occlusion (MCAO). Rats were randomly divided into various groups as described following: 1) Vehicle + MCAO; 2) Vitamin C + MCAO (Vitamin C + MCAO served as positive control); 3)-5) Tomato + MCAO (T2, T10 and T50 + MCAO). The animals in group 2) were treated with Vitamin C (250 mg/kg BW), a well known antioxidant which previously reported to exert the neuroprotective effect. The animals in group 3)-5) were treated with tomato pomace powder at doses of 2, 10 and 50 mg/kg BW respectively. The dosage range of tomato pomace powder used in this study was selected based on the previous data about the effect of tomato on the central nervous system and Vitamin C was used as positive control because it has been previously demonstrated to exert the neuroprotective effect. All administered substances used in this study were freshly prepared and used propylene glycol as vehicle.

All rats were fasted for 12 hr but they were allowed free access to water before surgery. Anesthesia was induced with intraperitoneal injection of thiopental sodium at dose of 50 mg/kg BW. Focal cerebral ischemia was induced by modified previous method described by Longa et al. Briefly, the right common carotid artery and the right external carotid artery were exposed through a ventral midline neck incision and were ligated proximally. A silicone coated nylon monofilament (4-0) suture (USS DG^TM sutures; Tyco Healthcare group LP, Connecticut, USA) with its tip rounded by heating near a flame was inserted through an arteriectomy in the common carotid artery just below the carotid bifurcation, and then advanced into the internal carotid artery approximately 17 to 18 mm distal to the carotid bifurcation until a mild resistance was felt. Occlusion of the origins of the anterior cerebral artery, the middle cerebral artery, and the posterior communicating artery was thereby achieved. Then, the wound was sutured, the rats were returned to their cages with free access to food and water. The incision sites were infiltrated with 10% Povidone-Iodine Solution for anti-septic postoperative care.

All animals were killed 24 hours after occlusion, and the brain was removed and sectioned at 2-mm thick. Sections were immersed in 2% TTC (2,3,5-triphenyl tetrazolium chloride) for 30 minutes at 37˚C. Images of stained sections were digitized, and infarction volumes were determined using an indirect method.

In order to determine the activities of antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px), the ischemic brains tissues were weighed and homogenized with a buffer consisting of 10 mM sucrose, 10 mM Tris HCl, and 0.1 mM EDTA (pH 7.4), and then centrifuged at 3000 g for 15 min at 4˚C. The supernatant was used for bioassays. The activity of SOD was determined using a xanthine/xanthine oxidase system for production of superoxide radical and subsequent measurement of cytochrome c as a scavenger of the radicals. Optical density was determined using spectrometer (UV-1601, Shimadzu) at 550 nm. One unit of enzyme activity was defined as the quantity of SOD required to inhibit the rate of reduction of cytochrome c by 50%. SOD activity is presented as units per milligram of protein (U/mg protein). Catalase activity in the supernatant was measured by recording the rate of decrease in H₂O₂ absorbance at 240 nm. The activity of catalase was expressed as μmol H₂O₂/min/mg protein. GSH-Px was determined using t-butylhydroperoxide as a substrate. The optical density was spectrophotometrically recorded at 340 nm. One unit of the enzyme was defined as micromoles (µmol) of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidized per minute. The GSH-Px activity was expressed as U/mg protein.

Various brain brains of the rats including cerebral cortex, striatum and hippocampus were isolated and prepared as brain homogenate as mentioned earlier. Then, the level of lipid peroxidation in the brain homogenate was indirectly estimated by determining the accumulation of thiobarbituric acid reactive substances (TBARS).

Data were presented as mean ± standard error of mean (S.E.M.). The analysis was performed using one-way analysis of variance (ANOVA), followed by Dunnett’s test. All statistical results were considered significant if p-value < 0.05.

The amount of total phenol was determined with the Folin-Ciocalteu reagent. Gallic acid was used as a standard compound and the total phenols were expressed as mg of Gallic acid equivalent. Phenolic compounds had been regarded as antioxidant agents which acted as free radical terminators [10]. It was found that the dried tomato pomace powder used in this study contained total phenolic compound at the concentration of 413.07 ± 34.45 mg of gallic acid equivalent/mg.

Figure 1 showed that before the induction of cerebral ischemia, no significant changes of the neurological score. All groups still showed grade 5 of the neurological score. After the induction of neurological score, all treated groups showed the reduction of neurological score. At 7 days after the occlusion of right middle cerebral artery, rats which subjected to Vitamin C treated groups significantly enhanced the neurological score (p-value < 0.01)

compared to vehicle + MCAO treated group). When the treatment was prolonged further to 7 days after MCAO, all groups including both positive control and the tomato pomace treated groups showed the significant increase in neurological score (p-value < 0.01, compared to vehicle + MCAO treated group). At 14 days after MCAO, the significant increased neurological scores were still observed (p-value < 0.01 except Vitamin C treated group that showed p-value < 0.05, compared to vehicle + MCAO treated group).

We also assessed the infarct volume of various areas supplied by the middle cerebral artery. The results showed that rats subjected to Vitamin C treatment significantly decreased the infarct volume in hippocampus and striatum (p-value < 0.05 and 0.01 respectively; compared to the vehicle treated group) as shown in Figure 2. The rats which received tomato pomace powder at dose of 2 mg/kg BW could significantly decrease the infarct volume in both hippocampus and cerebral cortex (pvalue < 0.05 all; compared to the vehicle treated group). The increasing doses of tomato pomace powder to 10 and 50 mg/kg BW could produce significant reduction of brain infarct volume only in hippocampus (p-value < 0.01 all; compared to the vehicle + MCAO treated group).