Anticonvulsant and Sedative Activity of Tagara (Nymphoides macrospermum.)

Abstract

Tagara (Valeriana jatamansi. Jones) (Valerianaceae) is an important ayurvedic drug employed in several preparations used in the treatment of various diseases. In South India, a drug under the name Granthika tagara. (Kannada), botanically identified as Nymphoides macrospermum. Vasudevan (NM) (Menyanthaceae), is used in place of Tagara for the same ayurvedic preparations under the same formulations. The alcohol extract of N. macrospermum. was tested for its anticonvulsant and sedative activities. Alcohol extract of NM at doses of 500 and 750 mg/kg body weight, significantly reduced the severity and increased the latency of convulsions induced by pentylenetetrazole (PTZ) and also reduced the time taken for recovery. However, NM up to a dose of 750 mg/kg body weight did not exhibit any significant effect on pentobarbitone-induced hypnosis. The sedative effect of the plant extract was confirmed by a significant reduction in locomotor activity, when tested using an actophotometer. The results indicate that the plant extract exhibits anticonvulsant and sedative activities justifying its use as a substitute for V. jatamansi. in ayurvedic formulations.

Keywords:

• Actophotometer
• anticonvulsant
• Nymphoides macrospermum
• pentylenetetrazole

Introduction

Tagara is an important ayurvedic drug employed in several preparations used in the treatment of various illnesses such as epilepsy (apasmara.), anemia (pandu.), jaundice (kamala.), tuberculosis (yakshma.), mental disorders (unmadha.), fevers (jwara.), cough, and asthmatic conditions (swas-kas.) and also as a general and brain tonic (Mary et al., 1981). The accepted botanical source of Tagara is Valeriana jatamansi. Jones (Valerianaceae) (Anonymous, 1978). However, in South India, a drug under the name Granthika tagara. (Kannada), botanically identified as Nymphoides macrospermum. Vasudevan (Menyanthaceae), is used in place of Tagara (Yoganarasimhan et al., 1979). The genus Nymphoides. Hill consists of about 20 species, of which five are found in India; they are aquatic herbs, floating or creeping with white or yellow flowers (Santapau & Henry, 1976). N. macrospermum. is recorded as a new taxon from South India (Vasudevan, 1968). V. jatamansi. is reported to possess sedative and tranquilizer properties (Anonymous, 1976). Despite its traditional use, N. macrospermum. has not been subjected to detailed phytochemical or pharmacological studies (Rastogi & Mehrotra, 1993; Chatterjee & Pakrashi, 1995). The preliminary phytochemical analysis reveals the presence of steroid, tannins, saponins, and sugars (Mary et al., 1981). The anticonvulsant and sedative activity of V. jatamansi. has been carried out (Wagner et al., 1980), whereas no work has been carried out on N. macrospermum. (Iyengar, 1976; Mitra, 1985). The present work investigates the anticonvulsant and sedative effects of N. macrospermum. in mice.

Materials and Methods

Plant material

The root and rhizome constituting the drug of commerce of N. macrospermum. was procured from the crude drug market of Bangalore, Karnataka, India, during March 2005. The characters found in the market sample were pharmacobotanically analyzed and identified as N. macrospermum. (Yoganarasimhan et al., 1979; Mary et al., 1981). The material was also compared with the authentic herbarium specimen of N. macrospermum. Vasudevan (R. Vasudevan. 0966) deposited at the Regional Research Centre, Bangalore, India (RRCBI).

Preparation of extract

Dried and powdered roots and rhizomes (100 g) were macerated with 500 ml ethanol (90%). The extract was concentrated in a rotatory evaporator (Superfit, India); under reduced pressure to give a dry residue (15% w/w). The residue was dissolved in 1% Tween 80 for suitable concentrations.

Animals

Swiss albino mice of either sex weighing 25–30 g were housed in groups of six each in standard laboratory conditions at room temperature (22 ± 1°C) under a 12/12 h light/dark cycle. Animals were fed with commercial pellet chow with water ad libitum.. They were transferred to the laboratory at least 1 h before the experiment. All experiments were carried out in accordance with CPCSEA (Committee for the Purpose of Control and Supervision of Experiments on Animals) norms after obtaining approval from the Institutional Animal Ethics Committee, and each animal was tested only once.

Acute toxicity studies

Acute toxicity studies were carried out on Swiss albino mice by the method following Ghosh (2005). Alcohol extract at doses of 30, 100, 300, 1000, and 3000 mg/kg body weight were administered to separate groups of mice (n = 6) after overnight fasting. Subsequent to administration of drug extracts, the animals were observed closely for the first 3 h for any toxic manifestations, such as increased motor activity, salivation, clonic convulsions, and death, followed by observations at regular intervals for 24 h. The observations were continued for a further 7 days. At the end of the experimental period, the animals were observed for signs of toxicity, morphological behavior, and mortality (Jainu & Devi, 2004).

Anticonvulsant activity (PTZ-induced seizures)

The mice were divided into five groups of six animals each. The extract was orally administered to three different groups at doses of 250, 500, and 750 mg/kg body weight. The standard and control groups received diazepam (4 mg/kg, i.p.) and 1% Tween 80 (10 ml/kg, orally) respectively. PTZ was injected intraperitoneally into the extract-treated and control groups after 1 h and into the diazepam-treated group after 30 min. The animals were then individually placed in plastic boxes and observed immediately after PTZ injection for a period of 30 min. The latency and duration of myoclonic jerks as well as incidence of seizures were recorded (Hosseinzadeh & Madanifard, 2000; Kulkarni, 1981).

Pentobarbitone-induced hypnosis test

The mice were divided into five groups of six animals each. Four groups received the extract at doses of 250, 500, and 750 mg/kg and 1% Tween 80 (10 ml/kg) orally, respectively. The standard group received diazepam (3 mg/kg, i.p.). Pentobarbital sodium (45 mg/kg) was injected intraperitoneally after 1 h into the control and extract-treated groups and after 30 min into the diazepam-treated group. Loss of righting reflex was considered to occur when the animals remained on their back for 60 sec. This was noted as the onset of hypnosis. Duration of sleep was measured as the time interval between loss of righting reflex and the regain of righting reflex (Kulkarni & Kaul, 1980; Christina et al., 1982; Villar et al., 1992).

Locomotor activity

The effect of Nymphoides macrospermum. extract on locomotion was studied using an actophotometer (INCO, India), which consisted of a metallic box with a metallic activity cage (36 cm × 36 cm × 16 cm) provided with a grid floor. The equipment operated on four photocells connected in circuit with a counter. When a parallel beam of light falling on the photocell was cut off by an animal, a count was recorded on the counter. The animals were divided into five groups of six mice each. Locomotor activity was measured individually for each animal 1 h after oral administration of extract (250, 500, 750 mg/kg) and 1% Tween 80 (10 ml/kg) and 30 min after intraperitoneal injection of diazepam (3 mg/kg) as standard. The animals were individually placed in the center of the cage for a period of 10 min, and digital counts relating to locomotor activity were noted (Dews, 1953; Kulkarni, 1981; Kulkarni & Dandiya, 1975).

Statistical analysis

The data were expressed as mean values ± SEM and tested with one-way analysis of variance (ANOVA) followed by multiple comparison test of Dunnett.

Results

Acute toxicity studies revealed that N. macrospermum. extract was found to be nontoxic when administered orally to mice, and its LD₅₀ value was found to be higher than 3 g/kg. No lethality or any toxic reactions were found up to the end of the study.

In PTZ-induced seizures, the administration of N. macrosperum. extract, in a dose of 500 and 750 mg/kg1 h prior to the injection of PTZ, significantly (p < 0.01) delayed the onset of convulsions, and significantly (p < 0.01) shortened the time taken for recovery. Diazepam in a dose of 4 mg/kg totally abolished the episodes of convulsions. There was no significant effect of the extract in a dose of 250 mg/kg on the onset of convulsions and recovery (Table 1).

Table 1.. Effect of Nymphoides macrospermum. extract on PTZ-induced seizures, pentobarbitone-induced hypnosis, and locomotor activity in mice (Dunnett Multiple Comparison Test).

	PTZ-induced seizures		Pentobarbitone-induced hypnosis
Treatment	Onset of action	Duration of action	Onset of sleep (min)	Duration of sleep (min)	Locomotor activity
Control	48.33 ± 5.27	17.83 ± 1.54	5 ± 0.4	46 ± 3.42	410 ± 44.90
Diazepam	00 ± 00	00 ± 00	1.53 ± 0.1**	159.5 ± 5.32**	120.17 ± 31.56**
Extract 250 mg/kg	270.34 ± 5.16	14.02 ± 1.42	3.42 ± 0.75	69.61 ± 6.83	108.56 ± 26.83**
Extract 500 mg/kg	590 ± 168.72**	9.25 ± 1.34**	3.15 ± 1.05	67.23 ± 5.18	99.52 ± 22.08**
Extract 750 mg/kg	605 ± 177.36**	8.54 ± 1.78**	2.55 ± 0.95	66.48 ± 5.11	97.25 ± 21.12**

*p < 0.05; **p < 0.01.

Pretreatment of mice with the extract of N. macrosperum. in a dose of 250, 500, and 750 mg/kg 1 h before injection of pentobarbitone 45 mg/kg did not significantly increase the latency of hypnosis when compared with that of control. However, the duration of sleep was prolonged (p < 0.05). Diazepam in a dose of 3 mg/kg body weight half an hour prior to the injection of pentobarbitone significantly (p < 0.01) decreased the onset and increased the duration (Table 1).

N. macrosperum. in a dose of 250, 500, and 750 mg/kg significantly (p < 0.01) reduced the activity scores when measured in an actophotometer, in comparison with the control. Diazepam in a dose of 4 mg/kg body weight, half an hour prior to the measurement of activity, significantly (p < 0.01) decreased the activity scores (Table 1).

Discussion

N. macrospermum. exhibits anticonvulsant activity in the PTZ-induced seizure model. PTZ-induced seizure is analogous to petitmal type of seizures and human generalized seizures (Loscher & Schmid, 1988). Compounds effective against this experimentally induced seizure model are effective against the petitmal type of epilepsy (Vida, 1995).

The oral administration of N. macrospermum. at the dose of 500 and 750 mg/kg, 1 h before the injection of PTZ, delayed the onset of seizures and decreased the duration of seizures (Table 1). Drugs that are effective against petitmal seizures reduce T-type calcium currents, and these types of seizures can also be prevented by drugs that enhance GABA_A–BZD receptor-mediated neurotransmission, such as benzodiazepines and phenobarbitone (McDonald & Kelly, 1995). Studies have shown that activation of N.-methyl D-aspartate receptor (NMDA) is also involved in the initiation and generalization of PTZ-induced seizures (Nevis & Arnolde, 1989; Velisek et al., 1990). Drugs that block glutamatergic excitation mediated by NMDA receptors, such as felbamate, have an anticonvulsant property against PTZ-induced seizures (McDonald & Kelly, 1995). Anticonvulsant activity of N. macrospermum. may be attributed to one or more of the above mechanisms.

It may be inferred that N. macrospermum. did not have any potentiation effect on pentobarbitone-induced hypnosis at doses that exhibited anticonvulsant activity.

N. macrospermum. produced signs of CNS depression, indicated by a reduction in locomotor activity. This may be indicative of the probable impairment of motor neurons as a side effect of the anticonvulsant activity of N. macrospermum..

Conclusion

The alcohol extract of N. macrospermum. exhibited anticonvulsant and sedative activity. The result substantiates the traditional use of N. macrospermum. as a substitute for V. jatamansi. as an anticonvulsant and sedative drug.

Acknowledgments

The authors are grateful to the management of the M. S. Ramaiah College of Pharmacy, Bangalore, India, for financial support and help.