MATERIALS AND METHODS:

Animals:

Male Swiss albino mice (18-22 g) were divided into five groups consisting six mice in each as shown in Table 1. They were maintained at 25 ± 2°C and relative humidity of 45 to 55% and under standard environmental conditions (12 hrs light 12 hrs dark cycle). The animals had free access to food (Amrut feed, Chakan oil mills, Pune, India) and water ad libitum. All experiments were carried out between 12:00-16:00 hrs. The experimental protocols were approved by the Institutional Animal Ethical Committee (IAEC) of Bharati Vidyapeeth University, Poona College of Pharmacy, Pune- 411038.

Table 1: Drug treatment schedule for H. spicatum fractions

Groups	Treatment
I	1% Gum acacia
II	HSBF(03)
III	HSBF(10)
IV	HSBF(30)
V	Diazepam (1 mg/kg)

Preparation of n-butanol fractions of H. spicatum:

The 50 ml of n-butanol was added to the remaining dry powder of ethanolic extract of HS. This mixture was then shaken for 01 hour. The pressure was released intermittently. After 01 hour the mixture was allowed the settle for half an hour. The n-butanol layer was carefully decanted and collected in a china dish and evaporated to dryness to obtain n-butanol fraction of H. spicatum (HSBF).

Preparation of drug solution:

Accurately weighed quantity of dry powder of fractions was suspended in 1% w/v acacia mucilage to prepare the appropriate stock solution. Diazepam injection was diluted with the distilled water. The doses were administered by selecting the appropriate concentration of the stock solution.

Preliminary phytochemical and HPTLC screening^{3, 4}:

Preliminary phytochemical screenings of fractions were carried out using qualitative tests. The HPTLC system of CAMAG, Muttenz, Switzerland, Anchrome Enterprises (I) Pvt. Ltd, Mumbai, consisting of sample applicator (Linomat IV), Twin trough chambers with lid, UV viewing cabinet with dual wavelength (254/366 nm), HPTLC plates scanner III controlled by CATS software (version 4.06). The system of Anchrome Enterprises, Mumbai was used for HPTLC video documentation (CAMAG, Switzerland).

Sample was applied by the 100 micro-liter syringe (Hamilton) with help of sample applicator (Linomat IV) in the form of bands. Specifications were as follows (unless specified): Application position, 8 mm above the lower edge; start position, 10 mm; doses speed, 150nl/sec; band length, 8 mm; space between bands, 4 mm; quantity applied, 10 ml. The composition of mobile phase was 0.4% acetic acid: acetonitrile (gradient type- 0 minute - 0%, 3 minutes- 1.2%, 10 minutes -15%, 15 minutes -22%, 30 minutes -37.6%) for HS. Chromatograms developed from each band (the track) were observed in daylight and then in UV viewing cabinet with dual wavelength (254/366 nm). Plates were scanned in HPTLC plate scanner III controlled by software (version 4.06).

Acute toxicity study:

Healthy adult male albino mice (18-22 g) were subjected to acute toxicity studies as per guidelines (AOT 425) suggested by the Organization for Economic Co-Operation and Development⁵. The mice were administered with the different doses of n-butanol fractions of HS. The dose progression or reduction was carried out as suggested by the AOT-425 guidelines. The mice were observed continuously for 2 hrs for behavioral and autonomic profiles and for any sign of toxicity or mortality up to a period of 7 days.

Elevated plus maze (EPM):

Locally fabricated elevated plus maze consisting of two open arms (35 ´ 6 cm) and two enclosed arms (35 ´ 6 ´ 15 cm) was used. The maze was elevated to the height of 40 cm. Mice were placed individually in the center of the EPM facing an enclosed arm. The time spent by the mouse during the next 5 min on the open and enclosed arm was recorded. The animals received vehicle, and respective test drugs 60 min before and diazepam (1 mg /kg, i.p.) 30 min before their placement on the maze. Increased exploratory activity in the open arm was taken as an indication of anxiolytic activity^{6, 7}.

Double unit mirrored chamber test:

The mirrored chamber apparatus, fabricated locally, consisted of a mirrored cube (30´ 30´ 30 cm), open on one side and placed in square box. The container box (40´40´30 cm) had a white floor and black wall making 5 cm corridor, completely surrounding the mirrored chamber. A sixth mirror was placed on the wall of the box, positioned to face the open side of the mirrored chamber. The latency to enter the mirrored chamber and time spent in mirrored chamber during 5 min observation period was recorded 60 min after the respective drug administration. Diazepam (1mg/kg, i.p.) was used as a reference standard. The mice were not exposed to the apparatus before the test and evaluated only once to avoid habituation. The apparatus was washed after each evaluation to eliminate potential cues such excreta, urine left by the previous occupant^8,9.

Statistical analysis:

The comparison was made against the vehicle treated control group and the data was expressed as mean ± SEM. The data was analysed by one way ANOVA followed by post hoc Dunnett’s test using INSTAT software. The level of significance was p<0.05 (GRAPH PAD INSTAT, 2000).

RESULTS:

All animals treated with n-butanol fraction of H. spicatum (HSBF) were free of any toxicity as per acceptable range given by the OECD guidelines and no mortality was found upto 2000 mg/kg. Hence three different doses 3, 10 and 30 mg/kg were selected for further study. The preliminary phytochemical evaluation of HSBF showed presence of alkaloids, glycosides and saponin. The n-butanol fraction of H. spicatum extract showed only one band and physicochemical test confirmed the presence of saponin as shown in Table 2.

Table 2: The HPTLC analysis of n-butanol fraction Hedychium spicatum

Band number	Band number	Rf values	Nature of the phytochemicals
1	1	0.11	Saponins

Elevated plus maze (EPM):

On 2^nd and 7^th day of the treatment the vehicle treated control group showed the mean time spent in the closed arm as 215.87±15.24 and 244.48±13.09 seconds and in open arm as 36.88±9.472 and 26.22±2.87 seconds, respectively. The treatment with HSBF 03 mg/kg on 2^nd day and HSBF 03 and 10 mg/kg on 7^th day of the treatment significantly decreased the time spent in the closed arm and increased the time spent in open arm as compared to the control group on both the experimental days. HSBF 03 mg/kg on 2^nd day and HSBF 03 and 10 mg/kg on 7^th day of the treatment were equipotent in the time spent in the closed arm and open arm, whereas HSBF 10 mg/kg on 2^nd day was less potent in respect to the time spent in open arm to that of diazepam 1mg/kg. Diazepam 1 mg/kg was significantly decreased the time spent in the closed arm and increased the time spent in the open arm and thereby showed anxiolytic activity on both the experimental days (Table 3).

Table 3: Effect of HSBF on exploratory activities in EPM

Data expressed as mean ± SEM (n = 5). *p <0.05 and **p <0.01 as compared to control.

Time spent in closed arm (Seconds) (Mean ± SEM)
Treatments (mg/kg)	2^nd Day	7^th Day
Control (10 ml/kg)	215.87± 15.24	244.48± 13.09
HSBF 03	138.92± 11.61**	167.57± 12.48**
HSBF 10	184.55± 18.07	182.26± 11.46**
HSBF 30	208.96± 17.06	198.06± 17.54
Diazepam 1	110.88± 5.68**	105.52± 6.37**
Time spent in open arm (Seconds) (Mean ± SEM)
Control(10 ml/kg)	36.88± 9.472	26.22 ± 2.87
HSBF 03	76.30± 4.569**	86.58± 6.02**
HSBF 10	65.21± 5.896*	67.72± 9.99**
HSBF 30	36.16± 9.985	46.07± 6.78
Diazepam 1	78.17± 4.664**	84.38± 6.57**

Double unit mirrored chamber test:

In the vehicle treated control group the mean time spent in the closed arm as 104.366± 11.350 and 138.804± 13.513 seconds and the mean time spent in open arm as 40.028± 4.552 and 35.12± 4.348 seconds, on 2^nd and 7^th day of the treatment respectively. The treatment with HSBF 03 mg/kg on 2^nd day and HSBF 03 and 10 mg/kg on 7^th day of the treatment significantly decreased the latency to enter mirrored chamber and increased the time spent in mirrored chamber. Diazepam 1 mg/kg showed significant decrease in the latency to enter mirrored chamber and increase in time spent in mirrored chamber and thereby showed anxiolytic activity on both the experimental days. HSBF 03 mg/kg on 2^nd and 7^th day of the treatment was equipotent and HSBF 10 mg/kg on 7^th day of the treatment was less potent in the latency to enter mirrored chamber to that of diazepam 1mg/kg (Fig 1 and 2).

DISCUSSION:

The enhanced life expectancy coupled with stress and strain in today’s life resulted in increasing complaints of CNS¹⁰. These complaints are manifested from simple confusion to altered mental status which eventually may lead to loss of social life¹¹.In Indian traditional system of medicine, a large number of herbs are claimed to be useful in the various nervous complaints². Due to complex anatomy and physiology of CNS, the slight change in the drug concentration or its phytochemical can exhibit wide spread outcome which may further restrict its clinical utility¹⁰.

The elevated plus maze and double unit mirrored chamber are considered to be valid animal models of anxiety because both use natural stimuli (fear of a novel open space or multiple images and fear of balancing on a relatively narrow, raised platform) that can induce anxiety in humans¹⁰. The elevated plus maze is used to measure transfer latency period is markedly shortened if the animal had previously experienced entering open and closed arms, and this shortened transfer latency has been shown to be related with memory processes. Studies of several nootropics and amenestic agents on EPM made this model a widely accepted paradigm to study learning and memory processes in rodents¹². In present investigation, the n-butanol fraction of ethanolic extract of H. spicatum showed a significant nootropic potential in EPM and double unit mirrored chamber test (3 and 10 mg/kg) as compared to control, and is almost equipotent to that of diazepam. As reported earlier saponins well known for facilitation of learning and memory¹³. Various saponins like ginsenoside Rb 1 isolated from the plant Panax ginseng has been reported to promote cognitive performance in experimental animals^{14, 15}. The anti-anxiety action of the rhizomes of H. spicatum may due to presence of saponin. Hence, our studies for the first time confirm the traditional claim and further suggest the possible use of this plant in various behavioral and neurological complaints. A detail study is required to pin point the exact mechanism and molecular aspects of the nootropic potential of H. spicatum.

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Received on 21.07.2010

Accepted on 04.08.2010

Research Journal of Pharmacognosy and Phytochemistry. 2(5): Sept.-Oct. 2010, 403-406