Abstract: The present study was designed to investigate the sedative and cytotoxic activities of a crude methanol extract of leaves of Crataeva nurvala Buch-Ham. Sedative activity was evaluated by using hole cross, open field and Elevated-Plus Maze (EPM) tests at 400 mg kg-1 body weight. The crude extract decreased the locomotor activity of mice in hole cross, open field and EPM tests. The cytotoxic activity of this extract was determined by brine shrimp lethality bioassay where the LC50 value was found to be 55.46 μg mL-1 as compared to that of 0.451 μg mL-1 exhibited by standard vincristine sulphate. The result shows that the crude extract of the leaves of C. nurvala have significant (*p<0.05) sedative and cytotoxic activities.
INTRODUCTION
Crataeva nurvala Buch-Ham (synonym-Crataeva magna (Lour) DC; family-Capparidaceae), commonly known as Barna or Varuna (Bhattacharjee, 1998), is widely distributed throughout Bangladesh, India and tropical regions of the world (Kirtikar and Basu, 1984). It is a deciduous tree which grows upto 7-9 m in height. Traditionally it is useful as laxative, analgesic, antipyretic, anti-inflammatory, antihelminthic, antitumor, diuretic, demulcent, stomachic (Drury, 1978), rubefacient and febrifuge (Walia et al., 2007; Sanayaima et al., 2006). The previous phytochemical studies with C. nurvala led to the isolation of dodecanoic anhydride, methyl pentacosanoate (Gagandeep and Kalidhar, 2006), phragmalin triacetate (Haque et al., 2008), lupeol, friedelin, betulinic acid, sterols, saponins and diosgenin of which lupeol is reported to treat hypercrystalluria, hyperoxaluria and hypercalciuria (Anand et al., 1994). In the process of our continuous study with medicinal plants of Bangladesh (Kuddus et al., 2012; Al Amin et al., 2012), the present study has been undertaken to evaluate the sedative activity and cytotoxicity of C. nurvala as well as to find out the logical evidence for its folk uses.
MATERIALS AND METHODS
Collection of plant materials: Leaves of C. nurvala were collected from Chittagong University campus in April 2012 and identified by Dr. Sheikh Bokhtear Uddin, Associate Professor, Department of Botany, University of Chittagong, Bangladesh.
Animals: Swiss albino mice of both sex and weighing 32-37 g were collected from International Centre for Diarrheal Disease and Research, Bangladesh (ICDDR, B). The animals were housed in plastic cages having dimension of 28x22x13 cm under standard laboratory conditions (relative humidity 55-65%, room temperature 23.0±2.0°C and 12 h light: dark cycle) and acclimatized for 7 days and fed with standard diet and water. The ethical guidelines for the investigation of experimental animals were followed in all tests (Bowd, 1980) and the protocol was approved by the institutional committee.
Preparation of extract: The collected leaves were thoroughly washed with water, chopped, air dried for a week at 35-40°C and pulverized with an electric grinder. The powder obtained was extracted with methanol at room temperature for 7 days with occasional shaking and stirring. The filtrate so obtained was concentrated to dryness by evaporation of solvent using a rotary evaporator under reduced temperature and pressure.
Assay for sedative activity
Open field test: This experiment was carried out according to published
method (Gupta et al., 1971) where the floor of
an open field of half square meter was divided into a series of squares each
alternatively colored black and white. The apparatus had a wall of 40 cm high.
The number of squares visited by the mice was counted for 3 min, on 0, 30, 60,
90 and 120 min during the study period.
Hole cross test: This test was done for CNS depressant activity in mice (Takagi et al., 1971). The animals were divided into three groups-negative control, positive control and test animals. The test groups received methanol extract of C. nurvala leaves at 400 mg kg-1 b. wt orally whereas the control group received vehicle (1% Tween 80 in water). A steel partition was made in the middle of a cage having a size of 30x20x14 cm. A hole of 3 cm diameter was made at a height of 7.5 cm in the center of the cage. The total number of passages of a mouse through the hole from one chamber to another was counted for a period of 3 min on 0, 30, 60, 90 and 120 min after the oral administration with test substances. In this test, diazepam (1 mg kg-1 b.wt.) was used as the positive control.
Elevated plus maze (EPM) test: This experiment was performed by the method, the experimental details of which could be found elsewhere (Lister, 1987). It utilizes an equipment consisted of two open arms (5x10 cm) and two closed arms (5x10xl5 cm) radiating from a platform (5x5 cm) to give the apparatus a plus sign in appearance. The apparatus was situated 40 cm above the floor in which the open arms edges were 0.5 cm in height to keep the mice from falling and the closed-arms edges were 15 cm in height. The maze floor and walls were made with dark opaque wood. Sixteen minutes after administration of the test agents, each animal was placed at the center of the maze facing one of the enclosed arms. During the five min test period, the number of open arms entries was recorded. The entry into an arm was defined as the point when the animal places all four paws onto the arm. This procedure was conducted in a sound free room and observations made from an adjacent corner.
Cytotoxic activity: The cytotoxicity assay was performed on brine shrimp nauplii by standard method (Meyer et al., 1982) using vincristine sulphate as standard. The test sample was prepared by dissolving in dimethyl sulfoxide solution, DMSO (not more than 50 μL in 5 ml solution) plus sea water (3.8% NaCl in water) to attain concentrations of 25, 50, 100, 200, 400 and 800 μg mL-1. A vial containing 50 μL DMSO diluted to 5 mL-1 with sea water was used as negative control. Then matured shrimps were applied to each of all experimental and control vials. After 24 h, the vials were inspected using a magnifying glass and the number of survived nauplii in each vial was counted. From the data, the percent (%) of mortality of the brine shrimp nauplii was calculated for each concentration using the following formula:
where, Nt is Number of killed nauplii after 24 h of incubation, N0 is Number of total nauplii transferred i.e., 20. The median lethal concentration, LC50 was then determined using Probit analysis.
Statistical analysis: All data were expressed as mean±STD and were analyzed by one way ANOVA followed by using Dunnett’s test. The difference was considered significant at p<0.05.
RESULTS AND DISCUSSION
Sedative activity: Open field test: In this test, the number of squares traveled by the mice was suppressed significantly in the test group throughout the study period. The CNS depressant activity obtained for the extract was statistically significant (Table 1). About 30 and 90 min after the administration of the extract, the number of movements were 58.33 and 11.0 per min as compared to that of 45.99 and 9.33 displayed by standard diazepam.
Hole cross test: The total number of hole crossed from one chamber to another by the mice was counted for control, standard and test groups (Table 2). In the hole cross test, the extract showed a decrease in locomotor activity in the test animals during observation period as evident by the reduction in number of hole crossed by the treated mice (number of movements after 120 min is 3.66 per minute) as compared to the control group. The result was comparable to the reference drug diazepam and was statistically significant (p<0.05).
Elevated plus maze (EPM) test: Result of EPM test is presented in Table 3. The extract at the dose of 400 mg kg-1 body weight, significantly decreased the percentage of entries of mice into the open arms as well as the percentage of time spent in the open arms of the EPM.
| Table 1: | CNS depressant activity of crude methanol extract of C. nurvala as determined by open field test in mice |
| Values are expressed as mean±SD (n = 5), CM: Crude methanolic extract of leaf of C. nurvala. One way Analysis of Variance (ANOVA) followed by Dunntt’s test. *p<0.05, significant compared to control | |
| Table 2: | CNS depressant activity of crude methanol extract of C. nurvala by hole cross test in mice |
| Values are expressed as mean±SD (n = 5); One way Analysis of Variance (ANOVA) followed by Dunntt’s test. *p<0.05, significant compared to control | |
| Table 3: | CNS depressant activity of methanolic extract of leaves of C. nurvala by elevated plus maze test in mice |
| Values are expressed as mean± SD (n = 5); One way Analysis of Variance (ANOVA) followed by Dunntt’s test. *p<0.05, significant compared to control | |
Now a day scientists are trying to produce low cost suitable medicines for the treatment of neurological disorders like seizure, anxiety, panic, agitation and insomnia. Gama- aminobutyric acid (GABA) is the major neurotransmitter in the brain which ends up producing an inhibitory effect on neurotransmitter release by binding with GABA receptor resulting hyperpolarization of the neuron and diminishing the chance of occurring a successful action potential. The pharmacological action of sedative drugs increases the effect of the GABA neurotransmitter by binding to the benzodiazepine site on the GABAA (via the constituent chlorine atom) leading to the depression of central nervous system (Riss et al., 2008). The study has examined some neuropharmacological activities of methanolic extract of C. nurvala. The plant extract was found to possess central nervous system depressant activity as indicated by the decrease in locomotor activity in mice in hole cross, open field and EPM tests. So, the methanolic extract of C. nurvala leaves may produce sedative action by hyperpolarization of the CNS via GABA receptor in the brain.
Cytotoxic activity: The brine shrimp lethality bioassay is rapid (24 h), simple (e.g., no aseptic techniques are required), easily mastered, inexpensive and requires small amounts of test material (2-20 mg or less). The bioassay has a good correlation with cytotoxic activity in some human solid tumors and with pesticidal activity. Since its introduction, this in vivo lethality test has been successively employed for providing a frontline screen that can be backed up by more specific and more sophisticated bioassays once the active compounds have been isolated.
The LC50 value of the extract was 55.46 μg mL-1 and that for standard vincristine sulphate was 0.451 μg mL-1 which showed. No mortality was found in the control group, using DMSO and sea water. The result of brine shrimp lethality bioassay was also promising. Test samples showed different mortality rate at different concentrations. The mortality rate of brine shrimp napulii was found to be increased with the increase in sample concentration.
CONCLUSION
In conclusion, we can say that Crataeva nurvala Buch-Ham contains chemical constituents having sedative and cytotoxic activity. This could provide a rationale for traditional uses of the plant as antitumor agent and suggests for further investigation and isolation of biologically active constituents responsible for the activity.