Animals
Male and female albino mice 25–30 g and Wistar rats weighing 150–210 g were obtained from a random bred colony in the animal house of Mashhad University of Medical Sciences. Animals were housed in colony room 12/12 hr light/dark cycle at 21 ± 2°C and had free access to water and food.
Plant material
Plants were collected from Torabat Hydarieh (in south of Khorassan province, I.R. of Iran) in October 1998 and dried in shadow and ground. The C. sativus L. was identified by Ferdowsi University (Ms. Molaei) and voucher samples were preserved for reference in the herbarium of School of Pharmacy, Mashhad, IR. Iran (143-0319-1).
Preparation of extracts
The powder of stigma or petal was extracted using maceration with ethanol or water. The powdered plant was macerated in water or ethanol (80 %, v/v) for 3 days and, subsequently, the mixture was filtered and concentrated under reduced pressure at 40°C. The yield (w/w) of aqueous and ethanolic extracts of stigma was 50.8% and 56.6%, respectively. The yield (w/w) of aqueous and ethanolic extracts of petal was 15.5% and 19%, respectively. The extracts were dissolved in normal saline.
Phytochemical screening
Phytochemical screening of the extract was performed using the following reagents and chemicals: Alkaloids with Dragendorffs reagent, flavonoids with the use of Mg and HCl; tannins with 1% gelatin and 10% NaCl solutions and saponins with ability to produce suds [24].
The maximum non-fatal dose (MNFD) and acute toxicity
Different doses of the extracts were injected to separate groups of five animals. After 48 h, the highest dose that did not induce any mortality was considered as the maximum non-fatal dose. The number of deaths was counted at 48 h after treatment. LD50 values and the corresponding confidence limits (CL, 95%) were determined by the Litchfield and Wilcoxon method (PHARM/PCS Version 4). Doses of 10, 40 (50), 70 and 100 % MNFD were chosen for most tests. Lower doses were found to be effective in the writhing test.
Antinociceptive study
Hot-plate test
The hot-plate test was assessed on groups of 8 mice. The temperature of a metal surface was maintained at 55 ± 0.2°C. Latency to a discomfort reaction (licking paws or jumping) was determined before and after drug administration. The cut-off time was 20 s. The latency was recorded before and 30, 60, 120, 150 and 240 min following intraperitoneal administration of the agents. The prolongation of the latency times compared with the values of the control was used for statistical comparison. Control received normal saline (10 ml/kg, i.p.) and morphine (10 mg/kg, i.p.) was used as reference drug [25].
Writhing test
Groups of 8 mice were used for controls and test mice. One hour after the administration of the extract, the mice were given an intraperitoneal injection of 0.7% v/v acetic acid solution (volume of injection 0.1 ml/10 g). The mice were placed individually into glass beakers and five min were allowed to elapse. The number of writhes produced in these animals was counted for 30 min. For scoring purposes, a writhe is indicated by stretching of the abdomen with simultaneous stretching of at least one hind limb. Control received normal saline (10 ml/kg, i.p.), diclofenac (10 mg/kg, i.p.) and morphine (10 mg/kg, i.p.) were used as reference drugs. Naloxone (2 mg/kg, s.c.) was administered 15 min prior to the extracts or morphine injections [25].
Anti-inflammatory study
Xylene-induced ear edema
Mice were divided into groups of seven. Thirty minutes after i.p. injection of the extract, diclofenac and dexamethasone, 0.03 ml of xylene was applied to the anterior and posterior surfaces of the right ear. The left ear was considered as control. Two hours after xylene application, mice were killed and both ears were removed. Circular sections were taken, using a cork borer with a diameter of 7 mm, and weighed. The increase in weight caused by the irritant was measured by subtracting the weight of the untreated left ear section from that of the treated right ear sections. The formula for computing percent inhibition was: average writhes in the control group (normal saline) minus writhes in the drug group divided by writhes in the control group times 100%. Control received normal saline (10 ml/kg, i.p.), diclofenac (10 mg/kg, i.p.) and dexamethasone (15 mg/kg, i.p.) were used as reference drugs [25].
Formalin induced inflammation
Rats were divided into groups of six. The inflammation was produced by subaponeurotic injection of 0.1 ml of 2% formaldehyde in the right hind paw of the rats on the first and third day. The animals were treated daily with the extracts or diclofenac intraperitoneally for 10 days. The daily changes in paw size were measured by wrapping a piece of cotton thread round the paw and measuring the circumference with a meter rule [26].
