Anticonvulsant and muscle relaxant activity of the ethanolic extract of stems of Dendrophthoe falcata (Linn. F.) in mice.

OBJECTIVE: To investigate the anticonvulsant and muscle relxant activity of ethanolic extract of stems of Dendrophthoe falcata in mice.
MATERIALS AND METHODS: The ethanolic extract of stems of D. falcata (100, 300 and 500 mg/kg, p.o.) was studied for its anticonvulsant effect on maximal electroshock-induced seizures and muscle relaxant activity at the same dose level using rota rod and traction test in mice.
RESULTS: Preliminary phytochemical analysis revealed presence of proteins, carbohydrates, glycosides, steroids, triterpenes, flavonoids, tannins and phenolic compounds. D. falcata ethanolic extract (DFEE) (100, 300 and 500 mg/kg, p.o.) significantly (P<0.001) inhibited seizures induced by MES, reduced the duration of Hind limb tonic extensor phase (HLTE) and a decline in motor coordination.
CONCLUSION: The ethanolic extract possesses anticonvulsant activity and muscle relaxant activity.

Introduction

Epilepsy is characterized predominantly by recurrent and unpredictable interruptions of normal brain function, called epileptic seizures. It is a variety of disorders reflecting underlying brain dysfunction that may result from different causes.[1] Although a number of antiepileptic drugs (AEDs) are available, seizures remain uncontrolled in more than 20% of the patients. Unfavorable responses including drug reactions or poor response to the presently available drugs also indicate a need for development of new drugs as alternatives.[2] Medicinal plants are believed to be an important source of new chemical substances with potential therapeutic effects. Several plants used for the treatment of epilepsy in different systems of traditional medicine have shown activity when tested in modern bioassays for the detection of anticonvulsant activity and many such plants are yet to be scientifically investigated.[3]

Dendrophthoe falcata (Linn. f.) belongs to family Loranthaceace and is known as ‘Vanda’ in the Indian Ayurvedic System of Medicine.[4] D. falcata is a large bushy usually glabrous branch-parasite and distributed more or less throughout India (about seven species found in India).The whole plant is used in indigenous system of medicine as cooling, bitter, astringent, aphrodisiac, narcotic and diuretic, and is useful in pulmonary tuberculosis, asthma, menstrual disorders, swelling wounds, ulcers, renal and vesical calculi and vitiated conditions of kapha and pitta.[56] D. falcata is reported to have antilithiatic, diuretic, cytotoxic, immunomodulatory activities[78] and wound healing, antimicrobial, antioxidant activities[9] and hepatoprotective activity.[10] In the traditional system of medicine D. falcata is recommended for the treatment of epilepsy.[11] The present study was undertaken to investigate anticonvulsant and muscle relaxant activity of ethanolic extract of stems of D. falcata (DFEE) in mice.

Materials and Methods

Plant Material

The fresh stems of D. falcata were collected during the month of September 2009 from district Barabanki, U.P. The plant material was authenticated by Dr. Tariq Husain (Scientist Herbarium), National Botanical Research Institute, Lucknow (reference no. 97307).

Drugs and Chemicals

All standard chemicals used in this study were of analytical grade. The drugs phenytoin was obtained from Sain Medicaments Pvt. Ltd., Hyderabad and diazepam from Helios Pharmaceutical Pvt. Ltd., BaddiThe plant extract, phenytoin and diazepam were dissolved in polyethylene glycol.

Preparation of Extract

Authenticated stems were air-dried, crushed to a moderately coarse powder. The powdered stems (approx. 175 g) was packed in soxhlet apparatus and continuously extracted with ethanol (95%) at 60-70° C till complete extraction. The solvent was removed by distillation and the concentrated extract was dried under reduced pressure at a temperature not exceeding 40° C in rotatory evaporator. A brown colour extract was obtained. The extractive yield was found to be 13.5%. The extract was kept in petri dish and stored in a desiccator at room temperature.

Preliminary Phytochemical Screening

A preliminary phytochemical screening of the extractsrevealed the presence of proteins, carbohydrates, glycosides, steroids, triterpenes, flavonoids, tannins and phenolic compounds.[1213]

Animals

Swiss albino mice (20-35 gm) of either sex (bred in D.R.D.O Gwalior, M.P.) were used. The animals were obtained from animal house of the Institute of Pharmacy, Bundelkhand University, Jhansi, India. The animals were housed in standard cages with free access of food (standard laboratory rodent's chow) and water. The animal's house temperature was maintained at 23 ± 3.0°C with a 12 hrs light / dark cycle. All the experimental procedures and protocols were reviewed by the Institutional Animal Ethics Committee (IAEC) of the Institute with reference no. BU / Pharm / IAEC / 09 / 010 (approved by CPCSEA Regd No. 716 / 02 / a / CPCSEA).

Acute Toxicity Study of the Extract

Acute toxicity studies were performed on Swiss albino mice according to Organization for Economic Co-operation and Development (OECD) – 425 guidelines.[14] The animals were kept fasted for 3 hours with free access to water. The ethanolic extract of stems of D. falcata was administered orally at a dose of 50 mg/kg. The dose at which mortality was observed in two out of three mice, it was considered as toxic dose. However, if no mortality was observed, the procedure was repeated with higher dose such as 100, 300, 500, 2000 and 5000 mg/kg body weight. Toxic manifestations like abnormal motor activity, alteration in water or food intake, respiration, sedation and moribund stages were observed for 6 h and mortality for 24 h. The LD50 was assessed in female Wistar rats and was found to be 4550 mg/kg.[15]

Locomotor Activity

Locomotor activity was studied using Actophotometer in five groups of five animals each. Group I received control, group II received diazepam (4 mg/kg), group III, IV and V received ethanolic extract orally at a dose of 100, 300 and 500 mg/kg, respectively. The locomotor activity for each animal was recorded for 5minutes at an interval of 2hrs.[16]

Maximal electroshock model

Maximal electroshock model was used to evaluate the anticonvulsant activity of extract. Corneal electrodes were used for bilateral delivery of electrical stimulus. Electroconvulsive shock (50 mA for 0.2 sec) was delivered through corneal electrode to induce Hind Limb Tonic Extensor (HLTE) phase in mice.[16] The mice were divided into five groups of five animals each (n=5).

Group I was treated with polyethylene glycol 10 ml/kg body weight.

Group II received phenytoin 25 mg/kg body weight.

Group III received oral 100 mg/kg of DFEE suspended in polyethylene glycol

Group IV received oral 300 mg/kg of DFEE suspended in polyethylene glycol

Group V received oral 500 mg/kg of DFEE suspended in polyethylene glycol

Rota rod test

The effect on motor co-ordination was assessed using Rota rod apparatus (Biocraft Scientific System Pvt. Ltd., Agra, India). The test was conducted on five groups of five mice each. Pretreated mice were trained to remain for 5 min. on the rod rotating at the speed of 25 rpm, The extract were administered 60 min. after the administration of ethanolic extract (100, 300 and 500 mg/kg) and 30 min. after the administration of diazepam (4mg/kg i.p.).[17]

Traction test

Forepaws of a mouse were placed on a 15 -cm long twisted wire rigidly supported and 20 cm above the table top. Normal mice grasped the wire with forepaws and when allowed to hang free, placed at least one hind foot on the wire within 5 sec. Inability to put up at least one hind foot was considered failure to the traction. The test was conducted on five groups previously screened mice, one hour after given extract (100, 300 and 500 mg/kg) and 30 min. after the injection of diazepam (4 mg/kg).[18]

Statistical analysis

Statistical analysis was done by one -way ANOVA followed by Dunnett's multiple comparison test vs. control. P<0.05 was considered significant.

Results

Preliminary phytochemical screening revealed the presence of proteins, carbohydrates, saponins, glycosides, steroids, triterpenes, flavonoids, tannins and phenolic compounds.

Acute Toxicity Study

During acute toxicity studies, DFEE (5000 mg/kg, p.o.) neither produced any abnormal effect - nor moribund stages. Moreover, no death was observed for next 14 days among these animals.

Anticonvulsant Assessment

Maximal electroshock produced hind limb tonic extension seizures in all the control animals. For duration of 15.22 ± 0.45 sec. DFEE at 100, 300 and 500 mg/kg significantly (P<0.01 and P<0.001) reduced the duration of the seizures as compared to control group. Phenytoin completely inhibited the MES-induced tonic seizures in all the animals [Table 1].

Table 1

Effect of D. falcata ethanolic extract (DFEE) on MES- induced seizures in mice. (n=5)

The ethanolic extract decreased the locomotor activity in mice at all doses [Table 2].

Table 2

Effect of D. falcata ethanolic extract (DFEE) on locomotor activity, rota rod and traction test

Muscle Relaxant Assessment

The mean time duration on rotating rod was significantly decreased (P<0.001) in animals treated with DFEE as compared to control group, indicating muscle relaxant activity and decline in motor coordination [Table 2].

Discussion

The maximal electroshock test is the most widely used animal model in evaluation of antiepileptic drugs. The present study revealed that ethanolic extract Dendrophthoe falcata attenuated MES induced tonic seizures indicating anticonvulsant activity. The MES test identifies agents with activity against generalized tonic clonic seizures using clinically established antiepileptic drugs. In addition to identifying drug activity against generalized tonic-clonic seizures, it has often been proposed that the maximal electroshock test predicts anticonvulsant drug effects against partial seizures. The anticonvulsant activity of ethanolic extract of Dendrophthoe falcata at 500 mg/kg in MES models indicates that DFEE stem might be precipitate the tonic-clonic seizures.

The present investigation also revealed that DFEE produced motor impairment and decreased spontaneous locomotor activity at anticonvulsant dose. This study has also been studied owing to its high rutin content which is responsible for anticonvulsant activity.[19] Various chemical constituents of plant origin, such as terpinoids, particularly triterpinoids and flavonoids, are reported to have muscle relaxant property[20] and anticonvulsant activity in various models of epilepsy like MES, PTZ, electrical kindling, etc.[21-25] Phytochemical screening of DFEE also showed the presence of flavonoids and triterpenes. Therefore it seems that the antiseizure and muscle relaxant activity of DFEE may be related to flavonoids and triterpenes present in extract.

Thus, it can be concluded that the ethanolic extract of D. falcata stem having significant anticonvulsant activity with muscle relaxant and decreased locomotor property. But the exact mechanism by which D. falcata exerts its anticonvulsant activity is not determined yet and needs further investigation to elucidate the other active compounds and underlying mechanism(s).