Evaluation of Anxiolytic and Anticonvulsant Effect of Benincasa hispida

 

Rachchh M.A.1*, Aghera R.R.1, Gokani R.H.1 and Jain S.M.2

1Department of Pharmacology, S. J. Thakkar Pharmacy College, Kalawad Road, Rajkot, Gujarat, India.

2Department of Pharmacology, L. M. College of Pharmacy, Navarangpura, Ahmedabad, Gujarat, India.

 

 

ABSTRACT:

 

Objective: Evaluation of anxiolytic and anticonvulsant effect of Benincasa hispida

 

Material and methods: The fresh fruit of Benincasa hispida was collected, air dried, seeds were separated and methanolic extract was prepared. Swiss albino mice of either sex, weighing between 30-40g were used. CNS activity of Methanolic extract of Benincasa hispida (MEBH) (300 mg/kg, p.o., 1000 mg/kg, p.o.) was measured by Open field behaviour model. Anxiolytic activity of MEBH (300 mg/kg, p.o.) was measured by Elevated plus maze (EPM) model using lorazepam (1 mg/kg, p.o.) as a reference standard. Anticonvulsant activity of MEBH (300 mg/kg, p.o., 1000 mg/kg, p.o.) was measured by Supramaximal Electric Shock induced convulsions using phenytoin (100 mg/kg, p.o.) as a reference standard. Skeletal muscles relaxant activity of MEBH (300 mg/kg, p.o.) was measured by Rota rod using lorazepam (1 mg/kg, p.o.) as a reference standard.

 

Result: In open field model MEBH showed increase in number of square crossed and number of rearing as compared to control group. In Elevated plus maze model MEBH showed significant increase in time spent in open arm and also increase the latency period for entry into close arm as compared to control group. In Maximal Electric Shock induced convulsions, MEBH showed significant reduction in duration of tonic flexion and tonic extension as compared to control group. MEBH showed the reduction in time spent on the rotating rod.

 

Conclusion: In short, MEBH showed mild CNS stimulant activity, significant anti-anxiety and anticonvulsant activity. MEBH lack muscle tone relaxation effect.

 

KEYWORDS: CNS stimulant, anxiolytic, anticonvulsant

 

INTRODUCTION:

Benincasa hispida (Thunb) Cogn. (Fam: Cucurbitaceae) is widely used as a vegetable in India and other tropical countries. It is commonly known as Bhuru Kolu or Safed Kolu (Gujarati), Petha (Hindi), White pumpkin (English) and Kushmanda (Sanskrit). Fruit of this plant is traditionally used as a laxative, diuretic, tonic, aphrodisiac, cardiotonic, urinary calculi, tridosha, blood disease, insanity, epilepsy, and also in cases of jaundice, dyspepsia, fever and menstrual disorder1. A well known sweet, ‘Agre ka Petha’, is formulated using its fruit pulp. It is also used in various Ayurvedic formulations such as Khanda Kooshmanda, Vasa Kushmanda Kanda, Kushmanda Ghrita etc2.

 

The methanolic extract of the fruit is reported to possess anti-ulcer3, anti-inflammatory4, antihistaminic5, anorectic6, nootropic7, anticonvulsant activity8 and antidepressant activity9. Phytochemical review indicates the presence of triterpenes: alnusenol, multiflorenol, iso-multiflorenol; flavone: iso-vitexin; Sterols: lupeol, lupeol acetate, and β – sitosterol10.

 

Since B. hispida plant is not investigated pharmacologically in detail, the present study is focused on the evaluation of the CNS activities (CNS stimulant, anxiolytic, anticonvulsant, Skeletal muscle relexant activities). Thus, this study may help us to explore and validate the use of B. hispida for various CNS disorders.

 

MATERIAL AND METHODS:

Animals:

Swiss albino mice of either sex, weighing between 30-40g were taken. Animals were fed with standard chow diet. All animals were kept under a controlled light/dark cycle and temperature (22 ± 2°C) with food and water ad libitum. Animals were divided into different groups. Coprophagy was prevented by fasting the animals in cages with grating on the floor. Throughout the experiment, the animal house was maintained in the same identical conditions. This experiment complied with the guidelines of our laboratory for animal experimentation and conducted after permission of IAEC.

 

Plant material:

The fresh fruit of B. hispida was collected in the month of September 2004 from the local vegetable market of Ahmedabad, Gujarat. The authentification of the plant was established and voucher specimen was deposited in the Department of Pharmacognosy and Phytochemistry, L. M. College of Pharmacy, Ahmedabad, India. The fruit was studied for its macroscopical and microscopical characters. The macroscopical characters and microscopical characters of the fruit were confirmed by comparing with the available literature. The fruit were air dried, seeds were separated and then dried mass was powdered using mixture. 100 gm of dried powder was defatted with petroleum ether and extracted with methanol (500ml × 5time) using soxhlate apparatus. Then methanolic extract was separate out using vacuum evaporator. Total yield of MEBH was 20% w/w. Extract was stored in a glass bottle in refrigerated condition throughout the period of experiment.

 

CNS activity using Open field behavior model:11

Three groups of mice were taken containing six animals in each group.

 

Group I:  Control: The animals received only aqueous suspension of 1% w/v sodium CMC as a vehicle (10 ml/kg, p.o.).

Group II:      Test-1:  Animals received MEBH (300 mg/kg, p.o.).

Group III:     Test-2:  Animals received MEBH (1000 mg/kg, p.o.).

This test was carried out in mice using the open field apparatus, which is a square box of 96 X 96 cm and about 15 cm high sidewall. The floor is divided into 16 equal squares. It was kept in a dimly lit and quite area during the experiment. Each mouse was placed at the same corner an hour after drug administration and allowed to explore the area for 5 minutes. The number of rearing and number of squares crossed were noted.

 

Anxiolytic activity using Elevated Plus Maze model:12

Three groups of mice were taken containing six animals in each group.

 

Group I:  Control: The animals received only aqueous suspension of 1% w/v Sodium CMC as a vehicle (10 ml/kg, p.o.).

Group II:             Test:            Animals received MEBH (300 mg/kg, p.o.).

Group III:   Standard Animals received lorazepam (1 mg/kg, p.o.) as a reference standard drug.

 

The apparatus used was a wooden ‘ plus’ shaped maze with two open arms and two closed tunnel like arms 25 X 5 cm and 36 cm high for mice and 50 X 10 cm and 40 cm high for rats. The maze elevated from the floor (25 cm for mice and 50 cm for rats). Test drugs were administered to the mice and after an hour they were placed individually on the centre square of the maze. For the next five minutes, the number of entries the animal makes to both open and closed arms and time spent there; the latency of first entry into the open tunnel and the entry preference were recorded. Each animal was used only once and the experiment was carried out at a fixed time of the day.

 

Test for muscle tone and balance by using Rotating Rod:13, 14

Three groups of mice were taken containing six animals in each group.

 

Group I:      Control:              The animals received only aqueous suspension of 1% w/v sodium CMC as a vehicle (10 ml/kg, p.o.).

Group II:    Test: Animals received methanolic extract (300 mg/kg, p.o.).

Group III:   Standard: Animals received lorazepam (1 mg/kg, p.o.) as a reference standard drug.

 

In this method the albino mice of either sex were used. Untreated mice were placed on a horizontal rotating iron rod having a diameter of 32mm and rotating at the rate of 24 RPM. Animals that remain on the rod for 2 or more minutes in four trials, carried out in two days divided into morning and evening two sessions, were selected and divided in to different groups. Vehicle and test drugs were administered by oral route and placed on rod at intervals for 4hr after the administration. The following parameters were noted down.

(i) Time of fall (in seconds)

(ii)  Percentage decrease in time of rotation

Anticonvulsant activity using Maximal Electric Shock model:15

Four groups of rats were taken containing six animals in each group.

 

Group I:      Control: The animals received only aqueous suspension of 1% w/v sodium   CMC as a vehicle (10 ml/kg, p.o.).

Group II:    Test-1: Animals received MEBH (300 mg/kg, p.o.).

Group III:   Test-2: Animals received MEBH (1000 mg/kg, p.o.).

Group IV:   Standard: Animals received phenytoin (100 mg/kg, p.o.) as a reference standard drug.

 

The test was carried out in four groups of rats. They were first tested for sensitivity to electric shock of 150mA. Only those rats, which exhibited full seizure components, were used for further experiment. The selected rats were given the electric shocks through corneal electrode wetted with normal saline. The current strength was 180mA for 0.2sec, delivered using INSIF electro convulsiometer. On next day again the drugs were given to all animals according to their groups and then after one hour, the shock of same intensity and duration stated above was given.

 

The following parameters were recorded.

·         Tonic flexion

·         Tonic Extension

·         Clonic seizure

·         Regaining of righting reflex.

·         Mortality, if any

 

The electric shock may produce the threshold discharge of neuro-transmitters in the brain areas (Exact site unknown), which may lead to tonic and clonic convulsions.

 

Statistical analysis:

The data obtained were analysed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple range test. P<0.05 was considered statistically significant.

 

RESULTS:

Here MEBH was tested at two different dose levels (300 mg/kg and 1 g/kg, p.o.) for CNS activity, anxiolytic, anticonvulsant and skeletal muscles relaxant activity.

 

CNS activity using Open field behavior model:

In open field behavior model, the generalized CNS activity of drug was measured in terms of increase in number of square crossed and increase in number of rearing. MEBH (300 mg/kg, p.o.) showed mild to moderate CNS stimulant activity, which was evident in the form of increased in number of square crossed and increase in number of rearing (77.78 ± 9.91,  64.11 ± 8.27), while with methanolic extract (1g/kg, p.o.), it showed (86.4 ± 14.06,  47 ± 7.55). Both these groups had higher values as compared to control group (68.22 ± 6.30, 40± 7.19) (Table 1).

Anxiolytic activity using Elevated Plus Maze model:

Here total time spent by the mouse in open arm (out of 300sec) was the indicator of the anxiolytic activity of the drug. Methanolic extract treated (172.5 ± 18.07) and standard Lorazepam treated group (244 ± 14.89) showed significant increase in total time spent in open arm (sec) as compared to control group (45.67 ± 9.04) (Table 2).

 

Sedative or tranquilizing effect using Rotating Rod:

The reduction in time spent on the rotating rod with methanolic extract at the dose level of 300 mg/kg (2.10%)  and at the dose level of 1g/kg (2.34%) were insignificant as compared to standard drug Lorazepam (97.45%) (Table 3).

 

Anticonvulsant effect Maximal Electric Shock model:

Methanolic extract at both the doses (300 mg/kg and 1g/kg, p.o.) showed significant reduction (p<0.05) in duration of tonic flexion (3.25 ± 1.31, 4.25 ± 0.63) as well as in duration of tonic extension (10.5 ± 0.96, 2.5 ± 0.5) as compared to tonic flexion and tonic extension seen in case of control group (7.5 ± 0.87, 12.5 ± 1.44). These values were also comparable with tonic flexion and tonic extension seen in case of standard, phenytoin (3.25 ± 0.48, 4 ± 0.91) (Table 4).

 

DISCUSSION:

In open field behavior test, the freezing time was significantly decreased at both dose levels. However, methanolic extract showed increase in number of rearing and number of crossing of squares. The observed results can be taken as a further evidence for the presence of mild CNS stimulant activity.

 

The MEBH showed significant antianxiety activity in elevated plus maze model, since the mice receiving treatments remained for a longer period of time in open arm as compared to the control group. The result was also comparable with that of standard drug Lorazepam.

 

Benzodiazepines are the widely use drugs for the treatment of anxiety. They produce their pharmacological action by binding to the site specific for them situated adjacent to the receptor of GABA. Among the other mechanisms involved in anxiolytic activity, one of the mechanisms is stimulation of 5–HT1A-receptor. This is an inhibitory auto-receptor that reduces the release of 5-HT and other mediators and causes anxiety suppression. Many other neurotransmitters and receptors have been implicated in anxiety and panic disorders16, particularly noradrenaline and neuropeptide such as cholecystokinin and substance P.

 

Some recent studies propose that the 5–HT receptor activity modulating groups have novel functioning as both anti-anxiety and antidepressants. Several partial agonists of 5HT1A receptors have been explored for potential utility both in anxiety disorders and in milder cases of mixed anxiety depression17. The 5-HT2C serotonin receptor is prominent in limbic forebrain and cerebral cortex.


Table 1: Effect of methanolic extract of Benincasa hispida on open field behavior

Group

Dose(p.o.)

Number of square crossed

Number of rearing

Control

10 ml/kg

68.22 ±  6.30

40.1 ±  7.19

Methanolic extract

300  mg/kg

77.78 ±  9.91

64.1 ±  8.27

Methanolic extract

1000  mg/kg

86.4 ± 14.06*

47.3 ± 7.55*

All values represent Mean ± SEM, n=6 in each group.

* p<0.05, when compared with the control group. (ANOVA, followed by Tukey’s multiple range test); F tab (2, 15) = 3.68; F cal = 0.67 (No. of square crossed); 2.03 (No. of rearing)

 

 

Table 2: Effect of methanolic extract of Benincasa hispida on elevated plus maze test in mice

Group

Dose

(p.o.)

Entry preference to open arm

Latency of first entry (sec)

Time spent in open arm (sec)

No. of entry in open arm

Control

10 ml/kg

2/6

0.98 ±  0.09

45.67 ±  9.04

7.83 ±  1.72

Methanolic extract

300 mg/kg

5/6

1.21 ±  0.09

172.5 ±  18.07*

8.0 ±  1.61

Lorazepam

1 mg/kg

6/6

2.14 ± 0.15*

244.0 ± 14.89*

3.2 ± 0.89

All values represent mean ± SEM, n=6 in each group.

* p<0.05, when compared with the control group. (ANOVA, followed by Tukey’s multiple range test); F tab (2, 15) = 3.68; F cal = 29.24 (Latency of first entry); 48.05 (Time spent in open arm); 3.51 (No. of entry in open arm)

 

 

Table 3: Effect of methanolic extract of Benincasa hispida on muscle tone and balance using Rota rod

Group

Dose (p.o.)

Fall off time Before (Sec)

Fall off time After (Sec)

% Decrease in time

Control

10 ml/kg

426.5 ± 59.17

417.5 ± 35.96

2.10

Methanolic extract

300 mg/ kg

320.0 ± 84.89

312.5 ± 81.40

2.34

Lorazepam

1 mg/kg

333.0 ± 82.21

8.5 ± 1.03*

97.45*

All values represent Mean ± SEM, n=6 in each group.

* p<0.05, when compared with the control group.  (ANOVA, followed by Tukey’s multiple range test); F tab (2, 15) = 3.68; F cal = 17.09 (% Decrease in time).

 

 

Table 4: Effect of methanolic extract of Benincasa hispida on supramaximal electric shock-induced convulsions in rats

Group

Dose (p.o.)

Tonic flexion (sec)

Tonic  xtension (sec)

Clonic  convulsion

(sec)

Regaining of righting reflex (sec)

Control

10 ml/kg

7.50 ± 0.87

12.50 ± 1.44

15.25 ± 2.36

151.3 ± 19.62

Methanolic extract

300 mg/kg

3.25 ± 1.31*

10.50 ± 0.96

 

17.00 ± 4.80

 

117.3 ± 3.04

 

Methanolic extract

1000 mg/kg

4.25 ± 0.63*

2.50 ± 0.50*

 

6.75 ± 1.11

 

96.3 ± 12.46*

 

Phenytoin

100 mg/kg

3.25 ± 0.48*

4.00 ± 0.91*

7.00 ± 2.12

50.0 ± 12.28*

All values represent Mean ± SEM, n=6 in each group.

* p<0.05, when compared with the control group.  (ANOVA, followed by Tukey’s multiple range test) F tab (3, 20) = 3.10; F cal = 5.24 (Tonic flexion); 23.32 (Tonic extension); 3.38 (clonic convulsion); 10.24 (Regaining of righting reflex)

 

 

 

 

This receptor subtype has been postulated to be a reasonable therapeutic target for depression or anxiety18. The possibility of test drugs acting on 5-HT receptors cannot be ruled out. The conformation of this possibility requires further studies since the formulations are not reported for such action.

 

The increased muscle tone is a common feature of anxiety states in humans. Thus the extract was tested for its effect on muscle coordination and balance by performing Rota-rod test in mice. Extract did not affect the time spent on rotating rod indicating lack of muscle tone relaxation effect. However increase in the free rides was observed.

 

The MEBH at both the dose level showed significant reduction in tonic flexion and tonic extension period, which was comparable with that of standard drug Phenytoin. The effect was also dose dependant. The effect of drug may be

 

 

attributed to the modulation of GABAergic circuit or may be due to the inhibition of NMDA receptor.

 

Epilepsy involves the inhibitory GABAergic system and the system of the excitatory amino acids glutamate and aspartate. Agents which reduce GABAA synaptic function and stimulate NMDA receptor provoke convulsions19.

 

CONCLUSION:

The present study revealed that Methanolic extract of B.hispida possesses CNS stimulant activity, anxiolytic activity and anticonvulsant activity, while it is devoid of skeletal muscle relaxant activity. These activities are due to triterpenes, flavones and sterols presents in it. Further work is going on to isolate and identify responsible phytoconstituents for said activities.

 

 

REFERENCES:

1.        Kirtikar KR, Basu BD. Benincasa hispida. In: Blatter E, Caius JF, Mhaskar KS ed. Indian Medicinal Plants, 2nd ed., vol II. Dehradun: M/s Bishen Singh Mahendra Palsingh, 1975:1126-1128.

2.        Nadkarni KM. Benincasa hispida. In: Nadkarni KA ed. Indian   Materia Medica, 3rd ed., vol I. Bombay: Popular Book Depot, 1954;185-186.

3.        Grover JK, Adiga G, Vats V, Rathi SS. Extracts of Benincas hispida prevent development of abcdexperimental ulcers. J Ethnopharmacol 2001;78(2-3):159-164.

4.        Chandrababu S, Umamaheshwari S. Studies on the anti-inflammatory activity of fruit rind extract of Benincasa hispida Cogn. Indian Drugs 2002;39(12):651-653.

5.        Yoshizumi S, Murakami T, Kadoya M, Matsuda H, Yamahara J, Yoshikawa M. Medicinal foodstuffs. Xi: histamine release inhibitors from wax gourd, the fruits of Benincasa hispida cogn. Yakugaku  zasshi 1998;118(5):188-192.

6.        Kumar A, Vimalavathini R. possible anorectic effect of methanol extract of Benincasa hispida (Thunb). Cogn, fruit. Indian J Pharmacol 2004;36(6):348-350.

7.        Kumar A, Nirmala V. Nootropic activity of methanol extract of Benincasa hispida fruit. Indian J Pharmacol 2004;36:130.

8.        Vyawahare NS, Khandelwal AR, Batra VR, Nikam AP. Herbal anticonvulsants. Journal of herbal medicine and toxicology 2007;1(1):9-14.

9.        Rukmani R, Nidhiya ISR, Suresh Nair, Anil Kumar. Investigation of anxiolytic-like effect of antidepressant activity of Benincasa hispida, methanol extract. Indian J Pharmacol 2004:129-130.

10.     Maiti SP, Roy RL, Bhattacharya TK. Chemical examination of ripe fruit of Benincasa hispida (Thunb). J Inst Chem. (India) 1992; 64(3):123-124.

11.     Bhattacharya et al. Manual of pre-conference workshop on research methodology in pharmacology 1993;3.

12.     Lister RG. Psychopharmacology 1987;92:180-185.

13.     Janseen PAJ, Jagencau A, Niemegeers CJE. J Pharm Exp Thera 1960a; 129:471.

14.     Dunham NW, Mija TS. J Am Pharm Ass Sci Ed 1957;46:208.

15.     Goodman LS, Grewal MS, Brown BC, Swinyard EA. J Pharm Exp Ther 1953; 108:168.

16.     Sandford JJ, Aggyropoulos SU, Natt DJ. The psychobiology of anxiolytic drugs-part I. Basic neurobiology Pharmacol Thera 2000; 88:1497-1952.

17.     Dubovsky SL, Buzan R. CNS Drugs 1995; 4:47-57.

18.     Murphy DL, Aulakh CS, Garick NA, Sunderland T. In: Meltzer HY ed, Psycho-pharmacology-The third generation of progress. New York: Raven Press, 1987:545-552.

19.     Goyal RK, Mehta AA, Balaraman R, Burande MD. Anti epileptic. In: Goyal RK edi. Derasari and Gandhi’s elements of pharmacology, 14th edition, Ahmadabad: B. S. Shah Prakashan 2005: 281.

 

 

Received on 09.08.2010

Accepted on 11.09.2010        

© A&V Publication all right reserved

Research Journal of Pharmacognosy  and Phytochemistry. 2(6): Nov. - Dec. 2010, 460-464