Activity of hydroethanolic extract from Kielmeyera coriacea stems on central nervous system in rats

The aim of the present study was to investigate psychotropic properties of the crude lyophilized hydroethanolic extract (HE) from Kielmeyera coriacea stems after acute or chronic administration by oral route (gavage) in rats. Anxiolytic, antidepressive and psychomotor stimulant effects were evaluated using Plus-Maze (PMT), Forced Swimming (FST) and Open Field (OFT) test experimental models, respectively. Evaluation of the extract stems (60.0 mg/kg) revealed decreased immobility time in the forced swimming test (FST), without increasing locomotor activity in OFT, after chronic administration. This effect of the extract was compared with imipramine (20.0 mg/kg). Acute administration of 120.0 mg/kg of extract stems increased the open arm entries percentage in the PMT. According to the results, Kielmeyera coriacea stems extract is effective as anxiolytic as well as an anti-depressive-like drugs.

Hypericum perforatum, a plant from the same family (Guttiferae) is considered an effective alternative in the treatment of mild to moderate depression (Muller et al., 1997;Josey and Tackett, 1999;Muruganandam et al., 2000).
The present work aimed to investigate whether the acute or chronic administration of the HE from Kielmeyera coriacea stems induces anxiolytic, antidepressant, or motor stimulant effects in rats.The experimental models chosen were the elevated plus maze (PMT), forced swimming (FST) and open field tests (OFT), respectively.

Plant material and extract
Kielmeyera coriacea was collected near Mogi-Guaçu (São Paulo, Brazil) in July, 1999.A voucher specimen (#SP298463) was deposited with the Herbarium of the São Paulo State Botanical Institute, São Paulo, Brazil.Species identification was performed by Dr. Maria Claudia Young of the same institution.
The dried and crushed stems (1.0 kg) of Kielmeyera coriacea were exhaustively extracted with 38 liters of ethanol/water (9:1) at room temperature for 7 days, yielding 167.3 g of extract after evaporation of the solvents and lyophilization.The resulting compound was registered by State University of Maringá under patent application # 001342 with the National Patents Institute (INPI) on October 9, 2002.

Animals
The experiment utilized male Wistar rats (50-55 days old, 220-250 g) provided by the Animal Housing, Facility of State University of Maringá, housed 5 per cage, in constant room temperature (22-23°C), under a 12-h light-dark cycle, with free access to food and water.

Treatment
The animals were treated once daily for one (acute treatment) or 40-45 (chronic treatment) day(s), with different doses of the HE from Kielmeyera coriacea stems administered by gavage in rats or the reference drugs, imipramine (20.0 mg/kg) or diazepam (2.0 mg/kg) by intraperitoneal (i.p.) route.HE from Kielmeyera coriacea stems and Imipramine HCl (20.0 mg/kg, Crystalia Chemical, i.p.) were dissolved in a vehicle that consisted of 0.9% NaCl (saline) containing 0.2% Tween 80. Diazepan was used directly from the ampoule in solution União Química.The procedures adopted were approved by the UEM Ethical Committee (# 084-02/COBEA) and followed according to the norms recommended by international guiding principles for Biomedical Research Involving Animals (CIMS), Geneva, 1985.

Forced swimming test (FST)
The FST was similar to that described by Porsolt et al. (1977;1978).Duration of immobility per a 5min period was measured.An animal was considered immobile when it ceased struggling and swimming, remaining floating in the water, making only necessary movements to keep its head above water.

Open-field test (OFT)
After 24 hours, each animal was placed in the OFT.During a 5min period, the number of squares visited (four feet placed in the same square) was registered using Royce's validation criteria (1977).

Elevated plus-maze test (PMT)
The procedure for the PMT was performed using the original method described by Pellow et al. (1986)

Chronic effect of HE from Kielmeyera coriacea stems on body weight increase and mortality
The same experimental groups used in the above experiments were assessed for body weight increase and mortality over the course of up to chronic treatment.After that period, the rats were killed and the organs were removed for observation and weighing.Body weight was measure every week.

Data Analysis
Results are expressed according to the mean ± SEM for each group.A one-way analysis of variance (Anova) followed by Dunnett´s test for multiple comparisons or the unpaired t-test was used.Effects or differences were considered significant at p=0.05.

Results
Acute administration of different doses of HE from Kielmeyera coriacea stems by gavage in rats did not affect any of the parameters analyzed in the FST or OFT when compared to control (saline + 2% Tween 80) treated group (data not shown).
Figure 1 shows the results observed after chronic administration (40-45 days) of HE from Kielmeyera coriacea stems (30.0 or 60.0 mg/kg) or imipramine (20.0 mg/kg) compared to control (Saline + 2% Tween 80) in rats submitted to the FST.The reference drug imipramine and 60.0 mg/kg of HE from Kielmeyera coriacea stems reduced immobility time significantly (F(3,24)=9.314,p<0.0003) compared to the control group in FST without an increase in the number of crossings in the OFT (F(3.24)=0.7623,p=0.5261).Table 1 shows effects of the chronic treatment with 60.0 and 120.0 mg/kg doses of HE extracts from Kielmeyera coriacea stems or diazepan (2.0 mg/kg) compared to control (saline + 2% Tween 80) on spatial/temporal and ethological parameters behavior in PMT in rats.The percentage of entries into open arms (F (3.29) = 16.7,p<0.0001) was increase by 120.0 mg/kg dose of HE from Kielmeyera coriacea stems (p<0.001).The time spent (F (3.29) = 20.94,p<0.0001) in open arms and the closed arm entries (F (3.39) = 1.76, p = 0.1751) was not altered significantly by different doses of the extract.The ethological parameters analyzed, risk-assessment (F (3.29) = 4.943, p=0.068) and head-dipping (F (3.29) = 17.73, p<0.0001) showed no significant effect with any dose of HE extracts from Kielmeyera coriacea stems.The reference drug, diazepan (2.0 mg/kg), increased significantly the spatio/temporal parameters, percentage of entries into (p<0.01)and time spent (p<0.001) in open arms, without alter the closed arm entries.The ethological parameters, riskassessment (p<0.01) and head-dipping (p<0.01) were significantly altered compared to control, revealing its anxiolytic effect.

Discussion
The present study showed that the HE from Kielmeyera coriacea stems was active orally and exhibits a significant, antidepressant-like effect in the FST model of depression in rats, only after chronic administration.
The therapeutic effect of antidepressant drugs after long-term use is known by popular medicine.Tricyclic or selective serotonin reuptake inhibitor antidepressant drugs shares the property of blocking serotonin (5HT), noradrenaline (NA) and/or dopamine (DA) transporters (Hyttel, 1994).This process takes place within a few hours, although the onset of the therapeutic effect ranges from 1 to 2 weeks of treatment, which is necessary for the development of specific adaptive changes (Blier and de Montigny, 1994, Harkin et al., 1999).A reduction in immobility time in the FST has been reported for the main classes of antidepressant drugs frequently after long-term administration (Porsolt et al., 1977(Porsolt et al., , 1978;;Borsine, 1995).
The results produced by HE from (2.0 mg/kg) Kielmeyera coriacea stems suggest that the increase in motor activity observed in the OFT is not related to the anti-immobility effect of the extract observed in FST, and confirms the specificity of their antidepressant-like effect.The efficacy of the 60.0 mg/kg dose of extract in reducing immobility time in the FST is comparable to the triclyclic antidepressive, imipramine, used as a reference drug.Imipramine significantly reduced immobility time without increasing locomotion.Acta Scientiarum.Health Sciences Maringá, v. 26, n. 2, p. 365-368, 2004 Anxiolytic effect of HE from Kielmeyera coriacea stems was observed by the percentage of entries into open arms parameter analyzed in PMT.The reference drug, diazepan, produced significant alteration in all traditional and ethological parameters.Nevertheless, the ethological analysis did not prove superior to traditional indexes (Cruz et al., 1994).
The behavior on the PMT is supposed to be driven by the conflict between fear (open space and novelty) and exploration (novelty).As a consequence, rats at elevated plus-maze tend to avoid the open arms and stay longer in enclosed arms.The percentage of open to total arm entries has been used as indexes of anxiety.Anxiolytic drugs increase the percentage of entries number onto and/or time spent in open arms, whereas anxiogenic drugs do the opposite (Pellow et al., 1986).Because changes in motor activity may also influence exploratory behavior in the PMT, the number of closed arm entries, that loads highly and exclusively on the factor associated with exploration, has been used to reflect this factor (Cruz et al., 1994).
Different doses of HE from Kielmeyera coriacea stems did not alter the number in closed arm entries in the PMT and did not increase the number of crossings in OFT, showing that the extract has no stimulant effect in CNS (Wash and Cummins, 1976).
In conclusion, our results suggest that the extract from Kielmeyera coriacea stems exhibits anxiolytic and antidepressant-like effects in the PMT and in FST in rats.Further studies are in progress to identify the active constituent and the mechanisms underlying the pharmacological activities observed.
. The number of entries and time spent in the open and closed arms of the maze were recorded for 5min.For statistical analyses, the percentage of open arm entries (100 x open/total entries) and time spent in open arms (100 x open/open + closed) and number of closed arm entries were calculated for each rat.