Rat Grouping and Garlic Administration
The Wistar rats, as well as SHRs, were randomly divided into three groups (6 male and 6 female rats in each group), i.e. (1) Group VI: rats receiving the administration of deionized water; (2) Group FG: rats receiving the administration of fresh garlic; (3) Group BG: rats receiving the administration of black garlic. In addition, the data obtained from Wistar rats and SHRs before the treatment was used as the control values, respectively. The flesh portions of fresh and black garlics were mashed to puree in a mortar. 100 mL of garlic homogenate with a concentration of 16% (w/v) were prepared by mixing 47.4 g of the fresh garlic puree (with a moisture content of 66.2%,w.b.) or 32.2 g of the black garlic puree (with a moisture content of 50.3%,w.b.) with deionized water, and stored at -10°C. The frozen garlic homogenates were thawed by tap water before being used. The deionized water, fresh garlic homogenate and black garlic homogenate were administrated to rats once a day at 10:00 AM for 14 days, using an injector equipped with an intragastric administration needle. The administration volume was 2 mL each time for a rat weighing 200-260 g, and 1.5 mL each time for a rat weighing 150-200 g. The average dose of garlic in dry matter was 1.38 g/kg rat. It corresponded to that an adult weighing 70 kg receives 10 g of dry garlic, based on the assumption that the dose for adults is one tenth of that for rats (Chen, 2006)
Measurement of the Body Weight, Blood Pressure and Heart Rate of Rats
Body weight was measured every three days using an electronic balance. Systolic blood pressure and heart rate were measured for the awake rats at 24 h before the first administration and 24 h after the last administration, with the caudal artery method (Li, 2002). The testing apparatus consisted of a tail sleeve, a pressure sensor, a bridge amplifier (QUAD bridge, AD Instruments, Australia) and a PowerLab data analytical processing system (8SP, AD Instruments, Australia). The measurement for each rat was in triplicate, and their average was used.
Biochemical Determinations for the Plasma and Hypothalamic PVN of Rats
The T-AOC and MDA content in plasma and PVN of rats were determined. In order to determine T-AOC and MDA content in plasma, 0.2 mL of blood was collected from each rat by tail venipuncture under the anesthetized condition with intraperitoneale (i. p.) injection of chloral hydrate (400 mg/kg), at 24 h before the first administration and 24 h after the last administration. Then, the blood samples were centrifuged at 3000 r/min and 4°C for 10 min. The plasma (supernatant) was separated and stored at - 80°C until being assayed. In order to determine TAOC and MDA content in PVN, all rats were sacrificed under the anesthetized condition with chloral hydrate (400 mg/kg, i. p.) at 24 h after the last administration. The brains were taken out quickly, and coronally cut in 500 μm thick slices using a cryostat. PVN (0.84 mm anterior and 0.60 mm posterior to the bregma) regions in the frozen slices were micro-dissected using a 26-gauge stainless steel tubing, and then stored at -80°C until being assayed. Before the assay, the frozen plasma was homogenized and thawed in ice water. The frozen PVN was homogenized by sonication with a pH 7.5 homogenization buffer (containing 140 mM NaCl, 5 mM KCl, 0.8 mM MgC12, l.8 mM CaC12, l mM Na2HPO4, 25 mM HEPES, l% glucose) in ice water, followed by centrifugation at 3000 r/min and 4°C for 10 min to attain the supernatant for the assay. For the plasma and PVN, the T-AOC was determined with the FRAP (ferric reducing ability of plasma) method (Benzie and Strain, 1996), and the MDA content was determined with the TBA (thiobarbituric acid) method (Placer et al., 1966). The soluble protein contained in PVN was measured with the Bradford method (Bradford, 1976). Commercial assay kits (Nanjing Jiancheng Bioengineer Institute, China) were used for the biochemical determination. T-AOC and MDA content in the plasma was expressed as U/mL and nmol/mL, while T-AOC and MDA content in the PVN was expressed as U/mg-protein and nmol/mgprotein, respectively. Data were obtained from each group of twelve rats throughout the experiments.
The group data were expressed as mean ± SD (standard deviation), and analyzed by one-way analysis of variance (ANOVA) using State 9.2 software (STATA Corporation, USA). Differences between the means were considered to be significant when p