Central Nervous System Depressant Activity of Ethanol Extract of Aerial Parts of Cynodon dactylon (L.) Pers. (Durva) in mice

Sharda Sonawane*, Deepak Bharati, Undale VR and Bhosale AV

Department of Pharmacology, PDEA’S SGRS College of Pharmacy, Saswad, Tal- Purandar, Dist- Pune, Maharashtra, India, 412301

ABSTRACT

Cynodon dactylon L. (Durva) a shrub from Poaceae family is popularly used in folk medicine for treating a wide variety of disorders in South and western India, China and central Asia.

Aim of the study: To investigate the CNS depressant properties on experimental animals.

Materials and methods: ethanol extract of aerial parts of Cynodon dactylon (EECD) was studied to investigate its CNS depressant pharmacological properties in the classical behavioral models (open-field, elevated plus maze-EPM, Rota-rod, and Barbiturate-induced sleeping time) using mice. We decided to use i.p. administration of drugs because this pathway allows faster viability of the ethanol extract of aerial parts of Cynodon dactylon than oral pathway using 50% propylene glycol as a solvent in mice at single doses of 50, 75 and 100mg/kg.

Results: No significant effect was evident on motor coordination of the animals in the rotarod test. On EPM, all the doses of EECD presented a significant reduction on the time of permanence in the open arms, indicating an absence of anxiolytic-like effect. In addition, the EECD increased the immobility time in the forced swimming test and potentiated pentobarbital-induced sleeping time in mice, confirmed a probable sedative and central depressant effect in the animals,

Conclusion: Our results suggest that the ethanol extract of c. dactylon at 75mg/kg and 100mg/kg dose biologically active substance(s) that might be acting in the CNS and have significant depressant and anticonvulsant potentials, supporting folk medicine use of this plant.

KEY WORDS: C. dactylon, central nervous system, EECD, EPM, FST.

INTRODUCTION:

A number of Indian medicinal plants have been used for thousands of years in the traditional system of medicine (Ayurveda). Cynodon dactylon (L.) Pers. (Family: Poaceae) commonly known as “Doob”, ”Durva” in India, is a weed and has been regarded to possess various medicinal properties.¹ The plant posses antimicrobial, and antiviral activity and has also been used to treat urinary tract infection, calculi and prostatitis. The aqueous plant extract is used as anti-inflammatory, anti-epileptic, diuretic, anti-emetic and purifying agent. It also has significant application in treating dysentery, dropsy and secondary syphilis.² Cynodon dactylon has been used as an Antidiabetic agent in traditional system of medicine in India. The many investigation reported the hypoglycemic, hypolipidemic and antihyperglycemic activity of the aqueous extract of Cynodon dactylon...

Although Cynodon Dactylon (Cyn. Dac) has been used in the folk medicine of many countries, however it is reported the plant effect against ischemia/reperfusion (I/R)-induced arrhythmias. In that study, probable anti-arrhythmic effect of Cyn.Dac was investigated in isolated rat hearts and finding showed marked protective effects of Cyn. Dac against I/R-induced arrhythmias in isolated rat hearts. Regarding the presence of flavonoid glycosides confirmed during phytochemical screening of the extract and their role in the scavenging of oxygen free

radicals, it seems one of the potential cardioprotective mechanism of the plant is anti-inflammatory properties. Probably, metabolic and/or direct mechanism(s) may also involve.³

It has been shown that flavonoid compounds present in various plants may produce beneficial effects in cardiovascular diseases such as atherosclerosis, coronary artery disease and hypertension⁴. It has also antioxidant properties ⁵. Diseases of the central nervous system implicate lifestyle. Non-genetic environmental factors, such as diet, emotional stress, social conflicts can produce CNS depression.

The objectives of the present study were to investigate CNS depressant effect of the ethanol extract of aerial parts of C dactylon and to determine whether the extract could possesses the CNS depressive action in experimental animals

EXPERIMENTAL:

Procurement of plant

Aerial parts of Cynodon dactylon (Durva) were obtained from regional parts of SGRS College of Pharmacy, Saswad. Obtained plant sample were collected, shade dried and send it to the Agharkar research institute, Pune for authentification.

Authentification of plant

Authentification of plant was done at Agharkar Research Institute, Pune; they have reported that given sample of plant is DURVA belongs to Cynodon dactylon (L.) Pers, Family-Poaceae.⁶

Preparation of the ethanol extract

Shade dried, powdered, sieved (40 mesh size) plant materials will be extracted first with petroleum ether (40-60OC) and then with ethanol. The ethanol extract will be evaporated to dryness. The trace amount of ethanol which might be present within the solid mass of extracts will be removed under vacuum.⁷

Drugs

Pentylenetetrazole (PTZ, 60.0 mg/kg, Sigma) was used as the convulsant drug; sodium Phenobarbital inj. (PB, 30.0 mg/kg) as the sleep-inducer drug. Diazepam inj. (DZP, 1.0 mg/kg) was used as a positive control in the plus-maze, Phenobarbital-induced hypnosis and Pentylenetetrazole-induced seizure tests. Imipramine hydrochloride (IMI, 15 mg/kg) was used as a positive control in the forced swimming test (FST). Propylene glycol solution was used as a vehicle. All treatments were administrated intraperitoneal (i.p.) pathway.

Animals

Male Wistar mice (30–35 g) were obtained from Serum Research Institute, Pune and maintained in our animal house under controlled temperature (23–25 ◦C) and 12 h light–dark cycle (lights on 07:00 h), with free access to food and water. All experiments were conducted in accordance with international standards of animal welfare recommended by the Society for Neuroscience . The experimental protocol was approved by the Institutional Research Committee. The minimum number of animals and duration of observation required to obtain consistent data were employed.

Elevated plus-maze test

The elevated plus-maze (EPM) test has been widely validated for measuring anxiolytic and anxiogenic-like activities in rodents⁸. This apparatus was made of Plexiglas and consisted of two open arms (30 cm×5 cm) and two closed arms (30 cm×5 cm) with 25 cm walls. The arms extended from a central platform (5 cm×5 cm). The maze was elevated 38.5 cm from the room’s floor. The mice were treated, 30 min before the test, with different doses of -EECD (50, 75, and 100 mg/kg, i.p.; n = 8), DZP (positive control, i.p.; n = 6) and Propylene glycol 50% (control group, i.p.; n = 6). Each animal was placed at the center of the maze, facing one of the enclosed arms. The number of entries and the time spent in enclosed and open arms were recorded for 5 min. Entry into an arm was defined as the animal placing all four paws onto the arm. Total exploratory activity (number of entries) and other ethologically derived measures (grooming, rearing, stretched attend postures and head dipping) were also registered. After each test, the maze was carefully cleaned up with a wet tissue paper (10% ethanol solution).

Forced swimming test

This test is the most widely used and recognized pharmacological model for assessing antidepressant activity⁹. The development of immobility when mice are placed in an inescapable cylinder filled with water reflects the cessation of persistent escape-directed behavior. The apparatus consisted of a clear Plexiglas cylinder (20 cm high×12 cm diameter) filled to 15 cm depth with water (24±1 ◦C). The mice were treated with different doses of EECD (50, 75 and 100 mg/kg, i.p.; n = 6), IMI (positive control, n = 6) and PG 2.5% (control group, n = 6). In the pre-test session, every animal was placed individually into the cylinder for 15 min, 24 h prior to the 5 min swimming test. During the test session the following behavioral responses were recorded by a trained observer: climbing behavior, which is defined as upward directed movements of the forepaws along the side of the swimming chamber; swimming behavior, defined as movement throughout the swimming chamber, which includes crossing into another quadrant; and immobility time, when the mice made no further attempts to escape, and makes only movements to keep its head above the water.

Phenobarbital-induced hypnosis

Sodium phenobarbital (a sub-hypnotic dose, 30.0 mg/kg) was injected i.p. 30 min after administration of EECD. The mice were treated with different doses of EECD (50, 75 and 100 mg/kg, i.p.; n = 6), the control group (n=6) was treated with PG solution and positive control group (n=6) was administrated with DZP 2.0 mg/kg in the same conditions. The effect was recorded for disappearance (latency) and reappearance (duration) of the righting reflex. Hypnotic sleeping time was considered to be the time interval between disappearance and reappearance of the righting reflex¹⁰.

Rotarod

The method of Dunham and Miya (1957) was used. The animals were placed with the four paws on a 2.5-cm diameter bar, 25 cm above the floor, which was turning at 12 rpm. For each animal, the number of falls (up to three falls) and the time of permanence on the bar for 1 min were registered.

Table -2 Effects of EECD on the forced swimming test in mice

Groups	Immobility time (sec)
Vehicle(50% PG)	76.5 ± 5.49
IMP (10mg/kg)	18.1 ± 2.74 ***
EECD(50mg/kg)	93.5 ± 9.15
EECD(75mg/kg)	106.2 ± 12.25 **
EECD(100mg/kg)	125.9 ± 10.11 ***

Each values represent mean ± S.E.M. **, ***P > 0.05 vs. controls (ANOVA and Student’s–Newman–Keuls test as the

post hoc test).

Table-3 Phenobarbital induced hypnosis

Groups	Latency (sec)	Duration (sec)
Vehicle(50% PG)	0	0
DZP(2mg/kg)	276.5 + 12.79**	2240.33 + 26.29**
EECD(50mg/kg)	483.83 + 21.16*	1921.16 + 25.16*
EECD(75mg/kg)	328.66 + 13.17**	2009.16 + 10.04**
EECD(100mg/kg)	219.16 + 9.13**	2621.33 + 69.91**

Each value is presented as mean ±S.E.M. (*) p < 0.05, (**) p < 0.01 as compared with the control group. (ANOVA one-way following by Dunnet test); n = 6 mice per group. Lat = latency, Dur = duration, DZP = diazepam, vehicle-50%PG

Fig. 1- Effect produced by different doses (50, 75, 100 mg/kg, i.p.) of EECD on the latency and duration of hypnosis induced by sodium pentobarbital (30 mg/kg, sub-hypnotic dose). The results are presented as mean ±S.E.M. (*) p < 0.05, (**) p < 0.01 as compared with the control group. (ANOVA one-way following by Dunnet test); n = 6 mice per group. Lat = latency, Dur = duration, DZP = diazepam, vehicle-50%PG.

Table-4 Effect of EECD on Rota-rod test in mice

Groups	Time of permanence (s)
Vehicle(50% PG)	175.53+1.38
DZP (2mg/kg)	37.16+0.87*
EECD(50mg/kg)	156.66+2.07
EECD(75mg/kg)	75.5+0.99**
EECD(100mg/kg)	40.33+0.71***

Each value represents mean + SEM for 6 no of animals.*, **, ***p ≤ 0.001 vs. control(ANNOVA and student’s- Newman-Keul’s test )

Statistics analysis

Data were analyzed by ANOVA for one-way and ANOVA and Student’s–Newman–Keuls test as the post hoc test

RESULTS:

EPM test

As shown in Table 1, EECD (75 and 100mg/kg, p.o.) decreased the NEOA (n◦ of entries in the open arms) and the TPOA (time of permanence in the open arms). Neither dose of EECD increased significantly the number of entries in the open arms, indicating an absence of anxiolytic effect. Diazepam (2 mg/kg, i.p.) treatment increased significantly the number of entries and the time of permanence in the open arms in 70.1% and 33.2% as compared with controls.

Forced swimming test

The animals treated with all doses of extract showed in Table 2,All animals treated with EECD showed no antidepressant behavior at all the doses (p > 0.05), The animals treated with imipramine (10 mg/kg, i.p.) as expected, decreased the immobility time in 76.3% as compared with control.

Phenobarbital-induced hypnosis

Since the Phenobarbital doses were sub-hypnotic the mice that received vehicle showed no changes in their behavior. Contrary, the animals treated with DZP as well as all animals treated with EECD at 75mg/kg and 100mg/kg doses evidenced a potentiation of the Phenobarbital hypnotic effect (Fig. 1). Hypnotic effect and the time to fall asleep did not show difference when compared to DZP group (p > 0.01).

Rotarod test

This test was performed to investigate whether the fractions were acting via the neuromuscular junction to mediate the observed effect rather than acting centrally. Alteration was observed on rotarod test after the treatment with both 75mg/kg and 100mg/kg doses of ethanol extract of C. dactylon L., as like diazepam (2 mg/kg, i.p.) decreased the time of permanence on the bar in this test as compared to controls showing myorelaxant properties as expected (Table 4).

DISCUSSION:

In this work, the effects of different doses of ethanol extract of aerial parts of Cynodon dactylon were studied in several behavioral animal models, as like as rotarod, elevated plus maze, barbiturate-induced sleeping time and forced swimming tests to evaluate possible central activity. We decided to use i.p. administration of drugs because this pathway allows faster viability of the ethanol extract of aerial parts of Cynodon dactylon than oral pathway. The tests cited above are classical animal models for screening of activities on central nervous system and providing information about psychomotor performance, myorelaxant, anxiety, and depressant activities. The acute treatment with the extract of aerial parts of C. dactylon roots did not present antianxiety effects in animal models of anxiety, but it seems to have interesting effect in depression models.

Fig -2- Effect produced by different doses (50, 75, 100 mg/kg, i.p.) of EECD on fall of time by rota-rod method

The elevated plus maze (EPM) test is the most popular test to search for new benzodiazepine-like anxiolytic agents¹². In this study, the EECD did not alter the performance of mice in the EPM test, suggesting that the extract, at 75 and 100 mg/kg doses used, did not interfere with anxiolytic activity. Diazepam, as expected, reduced the mouse’s natural aversion to the open arms, and promoted the maze exploration thereof.

It is known that decrease in sleep latency and increase in sleeping time is classically related to central nervous system depressant drugs¹⁰. It was found that the i.p. administration of the plant extract induced sedative effects in mice. Our results showed that the EECD in both doses decreased the sleeping latency time and increased the duration of sleep, suggesting a potentiation of Phenobarbital-induced sleeping time. However this test is not specific because compounds that interfere with biotransformation of Phenobarbital by cytochrome P-450 complex can show the same effects on central nervous system depressant drugs¹³.

On the basis of the clinical association of depressive episodes and stressful life events, many of the animal models for the evaluation of antidepressant drug activity assess stress-precipitated behaviors. The most widely used animal model for antidepressant screening is the forced swimming test. Although the relationship between immobility (a posture thought to reflect a state of “behavior despair” in which animals have given up the hope of scape) and depression remains controversial¹⁴, it is well demonstrated that drugs with antidepressant activity reduce the time during which the animals remain immobile¹⁵. In our results, a significantly increase in the immobility time for mice for all the doses of EECD was observed. In this way, the overall results seem to be predictive for depressant properties of the fractions.

Two doses of C. dactylon ( i.e. 75 and 100mg/kg) alter motor coordination in the rotarod test, as like diazepam (2 mg/kg), which decreased the time of permanence on the bar, suggesting that the actions of this plant, probably, may be exerted through peripheral neuromuscular blockage.

In conclusion, we showed that acute treatment with ethanol extract of aerial parts of Cynodon dactylon potentiated the barbiturate induced sleeping time and presents depressant effect as demonstrated in the EPM and forced swimming tests. Data obtained from rotarod method suggest that, suggesting that the actions of this plant, probably, may be exerted through peripheral neuromuscular blockage. The overall results confirm the popular use of this plant. Further studies are necessary to elucidate the pharmacological action of this plant.

ACKNOWLEDGEMENT:

Author is thankful to Poona District Education Association and SGRS College of Pharmacy, Saswad, for providing a platform to carry out this research work.

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Received on 24.05.2009

Accepted on 14.08.2009

Research Journal of Pharmacognosy and Phytochemistry. 1(2): Sept. - Oct. 2009, 119-122

Groups	No. of entries(n)		Time spent (s)
Groups	Open arms	Closed arms	Open arms	Closed arms
Vehicle(50% PG)	6.66+0.33	6.83+0.30	113.66+1.94	135.33+0.66
DZP (2mg/kg)	9.5+0.42*	6.5+0.42	170.33+1.78*	87.33+0.80*
EECD(50mg/kg)	8.16+0.30	7.5+0.22	93.16+1.66*	128.5+0.76
EECD(75mg/kg)	4.83+0.30**	6.16+0.30	72.83+0.79**	150.83+1.35**
EECD(100mg/kg)	3.16+0.30***	5.66+0.33	56.5+0.76***	183.33+1.22***