Plant Material of Cardiospermum halicacabum roots were collected during July-august from the villages in and around Bangalore. The plant material was authenticated from Department of Botany, Bangalore University.

Roots were dried at room temperature, crushed to powder and stored in a plastic container. The powdered material was successively extracted with petroleum ether and alcohol in soxhlet’s apparatus. Aqueous extract was prepared by maceration process with water for 72 hours. Each extract was concentrated by distilling off the solvent to obtain the crude extracts. The drug was extracted with each solvent till complete extraction is effected. The percentage yield of alcoholic and aqueous extract was found to be 12 and 2 % w/w respectively. Alcoholic and aqueous extracts were suspended in 4% gum acacia and were used to study the anxiolytic activity.

Swiss albino mice of either sex (18-20 g) procured from Bio. Need, Bangalore were maintained for 7 days in the animal house of P.E.S College of Pharmacy, Bangalore under standard conditions: temperature (24 ± 10 C), relative humidity (45-55%) and 12:12 light: dark cycle. The animals were fed with standard rat pellet and water ad libitum. The animals were allowed to acclimatize to laboratory conditions 48 h before the start of the experiment. Groups of 6 mice (18-24) were used in all sets of experiments. All the experiments were conducted after obtaining permission from the Institutional Animal Ethics Committee (IAEC) of PES College of Pharmacy, Bangalore.

Female, nulliparous and non pregnant mice weighing 18-22 g were fasted for 3 hours before administration of extracts. The dosing of the animals was done as per OECD guidelines [₁₃]. Number of animals died at the particular dose levels were recorded after 2days & 14 days of drug administration. LD₅₀ values were calculated using AOT 425 software provided by Environmental Protection Agency, USA

The plus-maze apparatus, consisting of two open arms (16 x 5 cm) and two closed arms (16 x 5 x 12 cm) having an open roof, was elevated (25cm) from the floor to observe anxiolytic behavior in mice. Mice were treated with vehicle (4% gum acacia, po)/diazepam (1 mg/kg, p.o)/ alcoholic extract of Cardiospermum halicacabum (ALECH), (100 or 300 mg/kg, p.o)/ aqueous extract of Cardiospermum halicacabum (AQECH), (100 or 300 mg/kg, p.o). One after the treatment, each mouse was placed at the center of the elevated plus maze with its head facing the open arm. During the 5 min experiment, following behavior of the mouse was recorded: 1) Number of entries in open and closed arms, 2) Total time spent in open and closed arm 3) Rearing and 4) Time spent in neutral zone. During the entire experiment, mice were allowed to socialize. Every precaution was taken to ensure that no external stimuli, other than the height of the plus-maze could invoke maze anxiety.

Light dark box is a rectangular box of 46 x 27 x 30 cm (l x b x h), which is divided in to 2 compartment 1/3 rd is for the dark compartment and 2/3 rd served as light compartment. Extract/vehicle or standard drug is administered through per oral route. Sixty min after oral administration the mouse are placed individually on the light compartment and observe for a period of 5 min. Number of rearing, number of locomotion, time spent in light and dark zones and number entries in light dark zone are observed during this observation period.

The open field apparatus is made up of plywood consists of 56 x 56 (l x b) cm. The entire apparatus is painted black and 6 mm thick white lines divided the floor in to 16 square of identical dimension. Open field is lightening by 40 W bulb focusing on to the field from the height of about 100 cm. The entire room, except the open field is kept dark during the experiment. One hour after the drug treatment, each animal were placed at one corner of the apparatus and the following behavioral aspects were noted in the next 5 min.

Latency: Time taken by animal to leave square in which it was placed

Ambulation: Number of square passed by animal

Rearing: Number of times animal stood on its hind legs.

Results were expressed as mean ± standard error of mean (SEM). Difference in means were compared using one way analysis of variance (ANOVA) followed by Dunnett’s test. P<0.05 were considered statistically significant.

Elevated plus maze (EPM): In the EPM, the behavior, which was observed, confirmed the anxiolytic activity of diazepam as reported previously.[₁₇] The ALECH and AQECH , at doses of 100 mg/kg, significantly (P < 0.001) increased the time spent in open arms and entries were increased at same dose. The ALECH and AQECH, at doses of 100 mg/kg and 300 mg/kg, significantly (P < 0.001) increased the time spent in neutral zone. ALECH, at doses of 100 mg/kg, significantly (P < 0.001) decrease the time spent in open arm (Table 1).

Figure 1

Table 1: Anxioltytic activity Alcoholic & Aqueous extract of C.halicacabum on Elevated Plus Maze Model

Values expressed are mean SEM, n = 6. P<0.05*, <0.01** and <0.001*** as compared to control group

Light-dark model: In the light dark model, the diazepam 1mg/kg significantly (P < 0.5) shown increase in latency and significantly (P < 0.001) increase time spent in light zone and in total locomotion at same doses. The ALECH, at doses of 100 mg/kg, significantly (P < 0.001) and the AQECH, at doses of 100 mg/kg, significantly (P < 0.01) decreased the time spent in dark zone, increased the time spent in light zone. The ALECH, at doses of 300 mg/kg, significantly (P < 0.05) decreased the time spent in dark zone and increase time spent in light zone. ALECH and AQECH at doses, 100 and 300 mg/kg, significantly (P < 0.001) increased the total locomotion (Table 2).

Figure 2

Table 2: Effect of ALECH & AQECH on Light-Dark Model

Values expressed are mean SEM, n = 6. P<0.05*, <0.01** and <0.001*** as compared to control group

Open field: In the open field diazepam (1 mg/kg), ALECH (100 mg/kg), AQECH (100 and 300 mg/kg), significantly (P < 0.01) increased total time spent in central compartment while ALECH (300 mg/kg), significantly (P < 0.05) increased total time spent in central compartment, no. of square crossed by animal (Table 3).

Figure 3

Table 3: Anxioltytic activity Alcoholic & Aqueous extract of C.halicacabum on Open Field Test

Values expressed are mean SEM, n = 6. P<0.05*, <0.01** and <0.001*** as compared to control group

On the eve of presenting this paper in internet journal of neurology, I take this as a unique opportunity to record my deep sense of gratitude to my esteemed research guide Dr. K. Nandakumar, Professor and Head, Department of Pharmacology, P.E.S. College of Pharmacy, Bangalore.

I am also thankful to Dr. Munniraju for his kind support for authentication and identification of this plant.