Effect of hydroalcoholic extract of Sphaeranthus indicus against experimentally induced anxiety, depression and convulsions in rodents

To investigate the effects of a hydroalcoholic extract of the Sphaeranthus indicus (SIE) against experimentally induced anxiety, depression and convulsions in rodents. The SIE (100, 200, 500 mg/kg, p.o.) was used in elevated plus maze, open field, forced swimming, and tail suspension tests in mice. The same doses were also used to evaluate its anticonvulsant effect on pentylenetetrazole (PTZ)-induced convulsions in mice and maximal electroshock (MES)-induced convulsions in rats. SIE was found to increase the number of entries and the time spent in the open arms of the maze at a dose of 100 mg/kg, p.o., indicating its anxiolytic activity. On the other hand, higher doses of SIE (200 and 500 mg/kg, p.o.) decreased open arm entries and time spent in the open arms of the maze in the elevated plus maze test indicating an absence of anxiolytic activity. However, this effect could have been related to a decrease in the locomotor activity of the mice and not to an anxiogenic effect, as indicated by the reduction in the total number of entries in the elevated plus maze. SIE also (at doses of 200 and 500 mg/kg, p.o.) decreased locomotor activity but did not affect emotional activity parameters in the open field test, suggesting a possible central nervous depressant activity. SIE also increased the immobility time in the forced swimming test at an oral dose of 500 mg/kg but did not significantly modify the activity in the tail suspension test. SIE protected rats against MES-induced convulsions and mice against PTZ-induced convulsions. Sphaeranthus indicus demonstrated anxiolytic, central nervous depressant, and anticonvulsant activities in rodents, thus supporting the folk medicinal use of this plant in nervous disorders.


INTRODUCTION
Sphaeranthus indicus Linn. (Asteraceae), commonly known as 'Gorakhmundi,' is a highly branched herb that is found to be distributed throughout the plains in India in wet places. In folk medicine, the entire herb is used for insanity, epileptic convulsions, vomiting, and hemicranias, [1] and is valued as an aphrodisiac and nervine tonic. [2,3] Previous phytochemical studies have reported the presence of a sesquiterpene lactone, [4] a steroid, [5] a fl avanoid, [6] an essential oil, [7] and eudesmanolides [4,8] in S. indicus. The herb has been reported to have antibacterial, antifungal, [9] immunomodulatory, [8] antioxidant, [10] and hypoglycemic [11] activities, while neuroleptic [12] and anxiolytic activity [13] has been reported for fl owers of this plant. These reported activities confi rm that the herb of S. indicus is able to modulate the physiology of the central nervous system. Various activities of the entire herb have been reported but no scientifi c data are available for the central nervous actions of the entire herb of S. indicus. Hence, the present study was designed to evaluate the effects of a hydroalcoholic extract of S. indicus on experimentally induced anxiety, depression and convulsions in rodents.

Animals
Albino Wistar mice (25-30 g) and rats (200-250 g) of either sex were bred in the Central Animal House facility of the institute and the animals were housed under standard conditions. Briefl y, they were maintained on a 12 h light/dark cycle and given free access to food and water up to the time of experimentation. The animals were acclimatized to the laboratory environment for one hour before the experiments. Animals were randomly distributed into groups of ten animals each. All experiments were conducted during the light period (08.00-16.00 h). All the protocols were approved by the Institutional Animal Ethics Committee (IAEC) and conducted according to the guidelines of CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals).

Plant material and preparation of extract
Fresh, fully grown flowering herbs of Sphaeranthus indicus were collected from the campus of our institute and authenticated by a taxonomist in the Department of Bioscience, Sardar Patel University, Vallabh Vidyanagar, Gujarat. A specimen of the plant has been kept in the herbarium of our institute (Voucher No. ARGH7). The plant material was completely dried under shade and powdered. The powdered material was extracted exhaustively with 50% ethanol by maceration for two days at room temperature with occasional shaking. The crude (hydroalcoholic) extract was fi ltered and dried under reduced pressure at 40ЊC (yield: 11.1% w/w).

Preliminary phytochemical screening
The hydroalcoholic extract of the Sphaeranthus indicus herb was tested for the presence of carbohydrates, proteins, alkaloids, flavonoids, glycosides, saponins, tannins, and essential oils using standard procedures. [14] Experimental protocol A freshly prepared aqueous solution of the dried extract of the S. indicus herb (SIE) was suitably diluted before administration to test animals. The vehicle, distilled water (10 mL/kg, p.o.), was administered to control animals. Diazepam was used as a reference drug for the elevated plus maze test (1 mg/ kg, i.p.), the open fi eld test (1 mg/kg, i.p.), and to study pentylenetetrazole-induced convulsions (4 mg/ kg, i.p.). Imipramine (10 mg/kg, i.p.) was used as a reference drug to study antidepressant action in the forced swimming and tail suspension tests. Phenytoin (25 mg/kg, i.p.) was used as a reference drug for maximal electroshock-induced convulsions. For the present experimental study, animals were divided into fi ve groups with each group consisting of ten animals. Group 1 served as a control group and received distilled water (vehicle; 10 mL/kg, p.o.), groups 2-4 served as test groups receiving SIE (100, 200, and 500 mg/kg, p.o) whereas group 5 served as a positive control and received the reference drug. The animals were subjected to various behavioral tests an hour after oral and 30 min after intraperitoneal administration.

Elevated plus maze test
The elevated plus maze used in this study was modifi ed from the one described by Lister (1987). [15] The plus maze consisted of two opposite arms, 25 cm ϫ 5 cm, crossed with two closed arms of the same dimensions with 30 cm-high walls. The arms were connected with a central square, 7.5 cm ϫ 7.5 cm, to give the apparatus the shape of a plus sign. The whole apparatus was elevated 25 cm above the fl oor in a dimly illuminated room. When exposed to this novel maze alley, the animals experienced an approach-avoidance conflict, which was stronger in the open arms than in the enclosed arms. Animals were placed individually in the center of the maze, facing a closed arm, after which the number of entries and time spent in the enclosed and open arms were recorded during the next fi ve minutes. An arm entry was defi ned as the presence of all four feet in that particular arm. A selective increase in open arm exploration was observed as a consequence of anxiolytic drug administration. The maze was cleaned after each trial to remove any residue or odor of the animals.

Open fi eld test
The apparatus consisted of a dimly lit area of 96 ϫ 96 cm that was divided into 16 squares. Mice were placed individually in one corner of the apparatus and observed for a period of three minutes to detect the number of peripheral squares crossed, number of central squares crossed, periods of immobility, as well as the numbers of rearings and fecal pallets. [16] Forced swimming test Mice were made to swim individually in a polypropylene vessel (30 ϫ 15 ϫ 30 cm) with a water level of 15 cm at 25 Ϯ 2°C. The mouse was initially allowed to swim for 10 min after which the total periods of immobility characterized by a complete cessation of swimming with the head just fl oating above water level, was determined during the subsequent fi ve-minute period. [17] Tail suspension test This test is a variant of the forced swimming test in which immobility was induced by suspending a mouse by its tail. Mice were hung on a wire individually in an upside-down posture so that their nostrils touched the water surface in a container. After initial vigorous movements, the mice assumed immobility during a fi ve-minutes observation period and these periods of immobility were recorded. [18] Maximal electroshock-induced convulsions Albino rats of either sex were given supramaximal electroshock of 150 mA for a period of 0.2 seconds through a pair of corneal electrodes by using an electroconvulsiometer (Techno., India). Animals showing positive hind limb extensor responses during prescreening were selected. These animals were treated as per the experimental protocols described above. The test was repeated after drug treatments on the next day. The rats were manually restrained and released immediately after the electrically induced convulsions and the seizure was observed throughout its course after stimulation. The severity of convulsions was assessed by the duration of tonic fl exion, tonic extensor, clonus, and stupor phase for each animal. The duration of each phase for each animal (sec) was measured by using a stopwatch. The criterion for anticonvulsant activity and protection against MES-induced seizures was the abolition of hind limb tonic extension (HLTE), which was taken as the end point of the test. [19] Pentylenetetrazole-induced convulsions The test was conducted in mice one hour after vehicle (1 mL/ kg, p.o.) or SIE (100, 200, and 500 mg/kg, p.o.) or diazepam (4 mg/kg, i.p.) treatment. Pentylenetetrazole (PTZ) was injected intraperitoneally (50 mg/kg) into groups of mice [20] which were observed for the incidence of convulsions, latency to fi rst convulsion and duration of convulsions.

Statistical analysis
The data were expressed as mean Ϯ S.E.M. Statistical analysis was done using one-way analysis of variance (ANOVA) followed by Dunnett's test. Results were considered signifi cant at P Ͻ 0.05.

Preliminary phytochemical screening
Phytochemical screening revealed the presence of carbohydrates, proteins, flavonoids, triterpenes, steroids, saponins, tannins, coumarins and essential oil in the hydroalcoholic extract of the S. indicus herb.

Effect on open fi eld test
The overall results of the open fi eld test are summarized in Table 2. As expected, when released into the open fi eld, the control animals started moving along the walls, with their initial exploration being limited mostly to the peripheral squares; the inner squares were rarely explored. Therefore, peripheral square entry in control animals was high and central square entry was low. Mice treated with a 100 mg/kg oral dose of SIE did not show signifi cantly altered peripheral square crossing, central square crossing, or immobility time   Table 3].

Effect on maximal electroshock-induced convulsions
The effects of SIE on the fl exion and extension phases of maximal electroshock-induced convulsions in rats are shown in Table 4. At the highest tested oral dose (500 mg/kg), SIE was found to signifi cantly decrease the duration of the hind limb tonic extensor phase in MES-induced seizures whereas the lower doses (100 and 200 mg/kg, p.o.) did not give any protection. Rats treated with phenytoin (25 mg/kg) were found to be completely protected from MES-induced convulsions, as demonstrated by the absence of all the phases of convulsion.

Effect on pentylenetetrazole-induced convulsions
All the oral doses (100, 200, and 500 mg/kg) of SIE were found to produce signifi cant (P Ͻ 0.05) and dose-dependent reduction in the duration of the fi rst clonic convulsion in mice [ Table 5]. However, SIE treatment (100, 200, and 500 mg/ kg, p.o.) did not signifi cantly affect the latency of onset of pentylenetetrazole-induced convulsions in mice; no change was observed in the incidence of convulsion and mortality. As expected, diazepam (4 mg/kg, i.p.)-treated mice did not have any convulsive episode or show any mortality when treated with pentylenetetrazole, the results demonstrated 100% protection of the test animals as compared to the control.

DISCUSSION
The present study evaluated the effects of a hydroalcoholic extract of the S. indicus herb on experimentally induced anxiety, depression, and convulsions. The elevated plus maze is considered to be an etiologically valid animal model of anxiety. The number of entries and time spent in the open arms have been found to be increased by anxiolytics and reduced by anxiogenic agents. [21] Anxiolytic activity was observed for an oral dose of 100 mg/kg dose of SIE. However, at higher oral doses of 200 and 500 mg/kg doses of SIE, signifi cant reduction in the number of entries and time spent in the open arms were observed. Furthermore, no anxiolytic or anxiogenic effects were observed with SIE treatment under these conditions as locomotor activity was impaired after SIE administration. Thus, there is a dose-dependency for the effects observed for SIE in this study. As expected, diazepam reduced the animal's natural aversion to the open arms and promoted maze exploration. Literature reports describe the action of benzodiazepines, such as diazepam, as anxiolytics  (at low doses) and as anticonvulsants, also producing sedation and myorelaxant effects at higher doses. [22,23] Reduction in the locomotor activity by the hydroalcoholic extract of S. indicus in the elevated plus maze test may be correlated with central nervous depression. [24] The open fi eld test is a paradigm used for evaluating the effect of drugs on gross general behavior and is used to measure the level of nervous excitability. [25] When removed from their acclimatized home cages and placed in a novel environment, animals express their anxiety and fear by showing decreased ambulation and exploration, immobilization or freezing, and reduction in normal rearing and grooming behavior. Increased micturation and defecation due to augmented autonomic activity is also observed. These paradigms are attenuated by classical anxiolytics and potentiated by anxiogenic agents. [16] The hydroalcoholic extract of S. indicus was seen to increase immobility time and decrease peripheral square movements; the observed decrease in central square movements could be due to impairment with locomotor activity. The decrease in locomotor activity in the open fi eld test of mice treated with hydoralcoholic extract of S. indicus, produces more evidence for its central nervous depressant activity. [24] Emotional activity parameters such as rearing and fecal dropping in mice were not signifi cantly affected by treatment with the hydroalcoholic extract of S. indicus.
Forced swimming and tail suspension tests are widely used to screen new antidepressant drugs. [17,26] The results of the forced swimming and tail suspension tests showed a signifi cant increase in the immobility time upon treatment with the hydroalcoholic extract of S. indicus. Thus, the overall results seem to be predictive for the central nervous depressant action of this hydroalcoholic extract of S. indicus.
The criterion for anticonvulsant activity and protection against maximal electroshock-induced convulsions was the abolition of hind limb tonic extension. Signifi cant reduction of hind limb tonic extensor phase in rats by prior administration of the hydroalcoholic extract of S. indicus (500 mg/kg, p.o.) may relate with its anticonvulsant action. Pentylenetetrazole is the most frequently used substance and an acute experimental model in the preliminary screening of potential anticonvulsant drugs. The induction of convulsions by pentylenetetrazole is attributed to the repression of the function of gammaaminobutyric acid type A (GABA A ) receptor Cl Ϫ channel. [27] Benzodiazepine agonists, like diazepam, are positive allosteric modulators of GABA-mediated neurotransmission in the central nervous system. Anticonvulsant potential of the hydroalcoholic extract of S. indicus was also demonstrated by the partial protection from pentylenetetrazole-induced convulsions.
The effi cacy of most herbal remedies is attributed to the combination of various active principles. The observed pharmacological actions of the hydroalcoholic extract of S. indicus may be due to the presence of a steroid, a saponin, a tannin, a fl avanoid, a coumarin, a triterpene, and an essential oil, as indicated by the results of preliminary phytochemical screening. As triterpenoids, [28] saponins, [29] fl avonoids, [28,29] and essential oil [30] from other plants have been reported to have central nervous system depressant activity, it is probable that the components that are present in abundance in the SIE might contribute, in part, for the observed central nervous system activity.

CONCLUSION
The results of the present investigation indicate that the hydroalcoholic extract of S. indicus has central nervous depressant activity. The investigation also highlights the fact that anxiolytic activity of this hydroalcoholic extract of S. indicus was observed only at a low dose (100 mg/kg, p.o.). Furthermore, the results obtained in the present study suggest that S. indicus has anticonvulsant activity, which lends pharmacological justifi cation to the use of the plant extract by traditional medicine practitioners in the treatment of epilepsy. Thus, this study provides experimental support for the traditional medicinal use of this plant for nervous disorders. Values are expressed as mean Ϯ SEM (n ϭ 10). One-way ANOVA followed by Dunnett's test, * P Ͻ 0.05 when compared with control group