ANTIDEPRESSANT EFFECT OF CROCUS SATIVUS L. STIGMA EXTRACTS AND THEIR CONSTITUENTS, CROCIN AND SAFRANAL, IN MICE

The effects of aqueous and ethanolic extracts of Crocus sativus L. stigma and their constituents safranal and crocin were studied for the antidepressant activity using forced swimming test in mice. The extracts and constituents were injected intraperitoneally to mice. The aqueous and ethanolic extracts of stigma (0.2-0.8 g/kg) decreased immobility time in comparison to normal saline. Safranal (0.15-0.5 ml kg) and crocin (50-600 mg/kg) also reduced immobility time. Swimming time was increased by fluoxetine and both extracts. Safranal increased swimming time. Climbing time was increased by imipramine and both extracts. Safranal with a higher dose (0.5 mg/kg) and crocin at doses 50 and 600 increased climbing time. In the open field activity test, the ethanolic extract and safranal increased stereotypic activities. On the basis of these results, the antidepressant effect of C. sativus stigma extracts may be mediated via safranal and crocin. Crocin may act via the uptake inhibition of dopamine and norepinephrine, and safranal via serotonin. INTRODUCTION Depression is a potentially life-threatening disorder that affects hundreds of millions of people all over the world. It can occur at any age from childhood to late life and is a tremendous cost to society as this disorder causes severe distress and disruption of life and, if left untreated, can be fatal (Bondy, 2002). The area of pharmacotherapy of depression started in the 1950s, with landmark publications and discoveries that still govern the manner in which we treat depression. There are currently between 10 and 20 different drugs marketed as antidepressants, depending on the country. Tricyclic antidepressants and monoamine oxidise inhibitors (MAOIs) were called first generation antidepressants, and selective serotonin reuptake inhibitors (SSRI) and reversible and selective inhibitors of monoamine oxidase A (RIMA) second generation antidepressants. Third-generation antidepressants include more recent molecules, such as mirtazapine, nefazodone, milnacipran, and reboxetine (Schulz and Macher, 2002). Some plants like Hypericum perforatum (Hippius, 1998; Franklin and Cowen, 2001, Schulz 2002), Curcuma longa (Ya et al., 2002) and Valeriana fauriei (Oshima et al., 1995) also showed antidepressant effects. Crocus sativus L. , commonly known as saffron, is a perennial stemless herb of the Iridaceae family, widely cultivated in Iran and other countries such as India and Greece. Commercial saffron comprises the dried red stigma with a small portion of the yellowish style attached. Compounds considered pharmacologically active and important are volatile agents (e.g. safranal), bitter principles (e.g. picrocrocin) and dye materials (e.g. crocetin and its glycosidic, crocin) (Ríos et al., 1996). Saffron extract or its active constituents, crocetin and crocin, could be useful as a treatment for neurodegenerative disorders accompanying memory impairment (Abe and Saito, 2002). Saffron is a protective agent against chromosomal damage (Premkumar et al., 2003), a potential cancer chemopreventive agent (Nair et al., 1991; Abdullaev, 2002), a modulator of lipid Proc. I IS on Saffron Eds: J.-A. Fernández & F. Abdullaev Acta Hort 650, ISHS 2004 436 peroxidation, and an antioxidant and detoxifying spice (Premkumar et al., 2003). Antinociceptive and anti-inflammatory (Hosseinzadeh and Younesi, 2002) as well as antiseizure (Hosseinzadeh and Khosravan, 2002) effects have also been reported in animals. Recently, we showed the antidepressant effect of C. sativus petal and stigma aqueous and ethanolic extracts in mice (Karimi et al, 2001). In this work, we studied the antidepressant effect and preliminary mechanism of C. sativus stigma and its two constituents, crocin and safranal in mice using modified forced swimming test. MATERIALS AND METHODS Animals Male BALB/c mice, weighing 22-25 g, were kept in the animal house of Mashhad University of Medical Sciences, in colony rooms with 12/12 h light/dark cycle at 21±2 ̊ C. The animals had free access to food and water. Preparation of Extracts In the maceration method, 4 g of stigma was macerated in 500 mL ethanol (80%, v/v) or distilled water for 24 h. The mixture was subsequently filtered and concentrated under reduced pressure at 50 ̊C. Forced Swimming Test The modified forced swim test was used for the evaluation of antidepressant activity in mice. Mice were placed individually in Pyrex cylinders (10 × 25 cm) that were filled with water at 24-25 °C to a 20-cm depth. They were removed 15 min later, dried and placed in their home cage. Twenty-four hours after their first exposure, the animals were replaced in the swim apparatus for 6 min and behaviours were monitored. Injections were administered intraperitoneally 30 min prior to the test session. After two min swimming, behavioural activities were evaluated during four min. Climbing behaviour consisted of upward directed movements of the forepaws along the side of the swim chamber. Swimming behaviour was defined as movement usually horizontal throughout the swim chamber, which also included crossing into another quadrant. Immobility was assigned when no additional activity was observed other than that required to keep the mice’s head above the water (Cryan and Lucki, 2000) Open Field Activity The apparatus, made of white wood, had a floor of 100 × 100 cm divided by red lines into 25 squares of 20 × 20 cm. The walls, 50-cm high, were also painted in white. The test room was illuminated at the same intensity as the colony room. Each mouse was placed in the centre of the open field, and its behaviour was observed for 10 min. The parameters evaluated were the total number of squares crossed. The numbers of leanings (one or two paws in contact with the wall), rearings (the mouse standing on its two hind paws without touching the walls), groomings (face cleaning, paw licking, fur licking, head scraping, and rubbing) were also recorded. At the end of each test, the whole area was cleaned with a wet sponge and a dry paper towel. Injections were administered intraperitoneally 30 min prior to the test session (Pardon et al., 2000). Statistical Analysis The data were expressed as mean ± SEM and tested with analysis of variance followed by the multiple comparison test of Tukey-Kramer.