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ORIGINAL ARTICLE
Year : 2014  |  Volume : 4  |  Issue : 2  |  Page : 81-87

Protective effect of leaf extract of Pandanus odoratissimus Linn on experimental model of epilepsy


1 Department of Pharmacology, JSPM's Jayawantrao Sawant College of Pharmacy and Research, Hadapsar, Pune, Maharashtra, India
2 Department of Pharmaceutical Medicinal Chemistry, Gahlot Institute of Pharmacy, Koparkhairane, Navi Mumbai, Maharashtra, India

Date of Submission08-Nov-2013
Date of Acceptance05-Jan-2014
Date of Web Publication29-Mar-2014

Correspondence Address:
Prafulla P Adkar
Department of Pharmacology, JSPM's Jaywantrao Sawant College of Pharmacy and Research, Handewadi Road, Hadapsar, Pune 411 028, Maharashtra
India
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Source of Support: JSPM’s Jayawantrao Sawant College of Pharmacy and Research, Hadapsar, Pune, Maharshtra., Conflict of Interest: None


DOI: 10.4103/2231-0738.129590

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   Abstract 

Objective: Study the anticonvulsant activity of ethanol extract Pandanus odoratissimus Linn in Swiss albino mice. Research Design and Methods: P. odoratissimus Linn (Pandanaceae) was evaluated for anticonvulsant activity in rodents. Animal models used include maximal electroshock test (MES); picrotoxin induced convulsions (PIC) and strychnine induced convulsion - (SIC). Results: Increase in latency to seizures as well as a reduction in duration and frequency of seizures indicated anticonvulsant activity. The selected extract was more effective in all models used except the SIC. P. odoratissimus ethanol extract (100 and 200 mg/kg body weight) significantly (P < 0.05-0.01) shortened the duration of convulsions in MES and PIC-induced seizures. Delay in the onset of convulsions in the two tests was significant (P < 0.01). Reduction in the frequency of seizures was also significant (P < 0.05, 0.01) in both tests. P. odoratissimus further delayed the onset of seizures in PIC induced seizures model while producing (66.7% and 83.33%) protection against death in mice. Diazepam (5 mg/kg) and marketed drug sample of P. odoratissimus (0.5 mg/kg) were used as reference anticonvulsant drugs for various models. Diazepam blocked the effect of the extract in the PIC and SIC tests significantly suggesting that P. odoratissimus may be acting by enhancing the effects of the gamma amino butyric acid ergic system. Conclusions: The ethanol extracts of P. odoratissimus Linn possess anticonvulsant activity since it delays the duration and the latency of seizures produced by MES and PIC.

Keywords: Anticonvulsant activity, anti-epileptics, ethanol extract, maximal electroshock, mice, Pandanus odoratissimus, seizures


How to cite this article:
Adkar PP, Jadhav PP, Ambavade SD, Shelke TT, Bhaskar VH. Protective effect of leaf extract of Pandanus odoratissimus Linn on experimental model of epilepsy. Int J Nutr Pharmacol Neurol Dis 2014;4:81-7

How to cite this URL:
Adkar PP, Jadhav PP, Ambavade SD, Shelke TT, Bhaskar VH. Protective effect of leaf extract of Pandanus odoratissimus Linn on experimental model of epilepsy. Int J Nutr Pharmacol Neurol Dis [serial online] 2014 [cited 2019 Nov 17];4:81-7. Available from: http://www.ijnpnd.com/text.asp?2014/4/2/81/129590


   Introduction Top


Epilepsy is a neurological disorder that affects a wide range of people throughout the world. It is a disorder of brain characterizes by unpredictable and periodic occurrence of a transient alteration of behavior due to the disordered, synchronous and rhythmic firing of populations of brain neurons .

It has been observed that the presently available antiepileptic drugs are do not provide cure nor prevent relapse and they are often associated with serious side-effects, including teratogenicity, chronic toxicity and adverse effects on cognition and behavior and unable to control seizures effectively in as many as 25% of the patients. [1],[2],[3]

Traditional system is believed to be an important source of chemical substances with potential therapeutic effects. Herbs may have antiepileptic effects in several ways. Some herbs may increase brain levels and/or the binding of nerve transmitter gamma amino butyric acid (GABA), which quiets nerve activity. [4]

Thus, it is necessary to investigate for an anti-epileptic agent that is highly efficacious as well as safe in items of drug related toxicity. The aim of treating an epileptic patient is not only to abolish the occurrence of seizures but also to lead a self-sustained life. [5]

Various classes of phytoconstituents such as alkaloids, lipids, terpenes, triterpenoids, flavonoids and coumarins have been reported to possess anticonvulsant activity. [6],[7],[8],[9]

Concurrently, phytochemicals identified from traditional medicinal plants are presenting an exciting opportunity for the development of new types of therapeutics. This has accelerated the global effort to harness and harvest those medicinal plants that bear substantial amount of potential phytochemicals showing multiple beneficial effects in convulsion. [10]

In the present study, we selected a plant namely Pandanus odoratissimus Linn belonging in to family Pandanaceae. It has forest habitat, it can reach a height of 150 cm. Kewda plants are found growing along seashores, banks of rivers, ponds, canals, etc., It grows in a tropical climate and wildly mainly in semi natural vegetation, where it can withstand with drought salty spray and strong wind. It propagates readily from seed but it is also propagated from branch cutting for farm or for garden. It grows fairly quickly. [7],[8],[9],[10]

Leaves are glaucous-green, 6-8 feet long, uniform, caudate acuminate, curvaceous, with spines on the margins and on the midrib. [11] The leaves are thought to be useful in leprosy, smallpox, scabies and diseases of the heart and brain. [12] Ayurvedic science has found the medicinal action of essential oil yielded by the screw pine's highly scented flowers to be useful in headaches, earaches and as a liniment for rheumatic pains.

It is also believed to have health-related properties, including antiviral, anti-allergy, antiplatelet, anti-inflammatory, Anti-diabetics, antioxidant and anticancer action. [13],[14],[15],[16],[17],[18],[19]


   Materials and Methods Top


Animals

Healthy Swiss Albino mice either sex weighing between 18 and 25 g chooses for the experimental animals were maintained in JSCOPR, Hadpsar, Pune animal house (12:12 dark: light cycle), with adequate ventilation, hygienic conditions, maintained on with normal pelleted diet [20],[21] (NUTRIVATE life science, Sinhagad Road, Pune, Baramati Agro Ltd. BVQI certified company) and water ad libitum. A group of animals were housed in polypropylene cage, paddy husk bed covered with stainless steel wire mesh with provision for water and feed. After obtaining prior permission from Institutional Animal Ethical Committee (IAEC) protocol approval no JSCOPR/IAEC/02/2012-13, all animals' studies were performed in accordance to guidelines of Committee for the Purpose of Control and Supervision of Experiments on Animals.

Plant material and extraction

The fresh leaves of P. odoratissimus Linn were collected and then washed with running water. The leaves were shade dried at room temperature and 1 kg of the dried leaf was ground into coarse powder. The powder was passed through a no. 60 mesh sieve. The ground powder was extracted then sequentially with Ethanol by using soxhlation method. The extracts were concentrated to dryness using Rotary Evaporator, (LABOROTA 4010/4011 digital) Heidolph Instruments GmbH and Co. KG Subsidiary: New Jersey, USA. The yield of the extracts was found to be 22.092% w/w (ethanol). [22],[23],[24]

Preliminary phytochemicals studies

The different extracts were then subjected to qualitative phytochemicals screening for the identification of the phytoconstituents. While petroleum ether, benzene, chloroform does not show any appreciable tests for the presence of different phytoconstituents, ethanol extract showed positive tests for the presence of glycosides, flavonoids and alkaloids. However, aqueous extract showed positive tests for glycosides and flavonoids only. The anticonvulsant activity of the ethanol extract of the plant in different dose levels (100 and 200 mg/kg) is being reported here. [25],[26]

Drugs and chemicals

Diazepam Injection: Calmpose, Ranbaxy Lab. Ltd., Batch no. 9021681 Badoi Dist. Solan. Mfg. Date: 05/2011, Exp. Date: 04/2014, was purchased from the local pharmacy and stock solution (2 mg/ml) for mouse test, was prepared freshly each day on the day of experimentation.

Diazepam (Calmpose ® ) injection was diluted with WFI to obtain a desired concentration of diazepam. Strychnine, Picrotoxin (Sigma-Aldrich®) Sigma chemicals, USA. [4],[5],[27],[28]

Acute toxicity studies

Acute oral toxicity study for the proprietary formulation was carried out using OECD guideline 425 (modified, adopted 23 rd march 2006).

The test procedure minimizes the number of animals required to estimate the oral acute toxicity of a chemical and in addition estimation of LD50, confidence intervals. The test also allows the observation of signs of toxicity and can also be used to identify chemicals that are likely to have low toxicity. [26]

The sequential test that uses a maximum of five animals, A test dose of 2000 or exceptionally 5000 mg/kg may be used in a situation where experiment has information indicating that the test material is likely to be non-toxic. [29]

As suggested, after acclimatization of animals for 4-5 days, study was carried out as follows.

Healthy, young adult Albino Swiss mice (18-25 g), nulli porous and non-pregnant were used for this study Food, but not water was withheld for 3-4 h and further 1-2 h post administration of sample under study.

Test dose was given to one animal by the oral route, this first test animal survived; four other animals were given the same dose (orally) as subsequent days and hence that a total of five animals were tested. Animals were observed individually at least every 5 min once during first 30 min after dosing, periodically at 2 h during the first 24 h (with special attention during the first 4 h) and daily thereafter, for a total of 14 days [Table 1].
Table 1: Observation of acute oral toxicity study


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   Experimental Design Top


Strychnine induced convulsion in mice

Swiss albino mice (18-25 g) were used for SIC in mice. The animals were divided in to five groups consisting of six animals in each group.

Group I was treated with Tween 80 (1%), Group II was taken as a standard and was treated with diazepam (5 mg/kg); group III and IV were taken as test group and treated with 100 and 200 mg/kg i.p. of the ethanol extract of leaves of P. odoratissimus Linn respectively.

The drug treatment was given continuously for 7 days (same dose daily). On 8 th day 60 min after extract administration strychnine was administered. The following parameters were recorded during the test session of initial, 30 min and up to 24 h. [4],[5],[30],[31]

  • Latency (onset of clonus)
  • Onset of tonic convulsions
  • Status of animal after 30 min
  • Status of animal after 24 h
  • Percentage protection.


Picrotoxin induced convulsion in mice

Swiss albino mice (18-25 g) of either sex were divided into five groups of six mice in each was fasted overnight prior to the test but the water was supplied ad libitum.

Group I was treated with Tween 80 (1%), Group II was taken as a standard and was with treated with diazepam (5 mg/kg). was treated with marketed preparation of P. odoratissimus Linn and Group III and V were served as test group and treated with 100 and 200 mg/kg i.p. of the ethanol extract of P. odoratissimus Linn respectively.

The drug treatment was given continuously for 7 days (Same dose daily). On 8 th day 60 min after extract administration PIC was administered. The following parameters were recorded during the test session of initial, 30 min and up to 24 h. [4],[5],[32]

  • Latency (onset of clonus)
  • Onset of tonic convulsions
  • Status of animal after 30 min
  • Status of animal after 24 h.


Maximal electroshock induced convulsion in mice

The Swiss albino mice (18-25 g) of either sex were divided in to five groups consisting of six animals in each group. Group I was treated with Tween 80 (1%); Group II was taken as a standard and was treated with diazepam (5 mg/kg). group III and IV were taken as test group and treated with 100 and 200 mg/kg i.p of the ethanol extract of P. odoratissimus Linn respectively. The drug treatment was given continuously for 7 days.(same dose daily) on 8 th day 60 min after administration 60 mA current was delivered transauricularly for 0.2 s in mice via small alligator clips attached to the cornea by using extracellular matrix and different phases of convulsion were recorded initial, 30 min and up to 24 h. [31],[32],[33]

  • Latency (onset of clonus)
  • Onset of tonic convulsions
  • Status of animal after 30 min
  • Status of animal after 24 h
  • Percentage protection.


Statistical analysis

All values were expressed as mean ± standard error of the mean. Result was analyzed statistically by using analysis of variance (ANOVA) followed by Dunnett's test. P ±0.05 were considered to be significant.


   Results Top


Acute oral toxicity study

Acute oral toxicity study was carried out in order to arrive at maximum tolerable dose of test formulation under study, according to modified OECD test guidelines 425. The test was restricted to limit test in view of no mortality being observed within 2000 and 5000 mg/kg of body weight. Daily thereafter, for total 14 days for sign of toxicity and/or mortality if any, The LD50 was calculated by using OECD 425 software. The details of study are tabulated and demonstrate in [Table 1].

SIC in mice

The marketed preparation and the ethanol extract of leaves of P. odoratissimus Linn were screened for antiepileptic activity in the SIC in mice. Chronic study was conducted using the 0.5 ml of marketed sample and the ethanol extract of plant, i.e., 100 and 200 mg/kg. The above mentioned doses were administered daily for seven consecutive days. It was observed that the dose of marketed sample and lower and higher doses (100 and 200 mg/kg) of plant extract did not produce significant anticonvulsant effect as compared to control. The standard drug diazepam (5 mg/kg) exhibited a significant anticonvulsant activity and offered 100% protection [Table 2] and [Figure 1].
Figure 1: Strychnine induced convulsion in mice. Group I: Control group (shows 100% mortality). Group II: #Diazepam (shows 100% Protection against convulsion). It does not produce any convulsion. Group III: Test control (marketed Sample) shows the shows 100% mortality but increase the onset of time of convulsion. Group IV: Test control 1 (ethanol extract of plant shows the 100% mortality but increase the onset of time of convulsion). Group V: Test control 2 (ethanol extract of plant shows the 100% mortality but increase the onset of time of convulsion)

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Table 2: Effect of ethanol extract of Pandanus odoratissimus Linn in strychnine induced convulsion in mice


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PIC induced convulsion in mice

The dose of marketed sample (0.5 ml) and the ethanol extract of leaves of P. odoratissimus Linn (100 and 200 mg/kg) in the PIC induced convulsion in mice shows the significant anticonvulsant activity. It shows the highest percentage protection 83.33% at the dose of 0.5 ml of marketed sample and 66.66% and 83.33% at the dose of 100 and 200 mg/kg of ethanol extract of plant [Table 3] and [Figure 2].
Figure 2: Picrotoxin induced convulsion in mice. Group I: Control group (shows 100% mortality). Group II: #Diazepam (shows 100% protection against convulsion). It does not produce any convulsion. Group III: Test control (marketed Sample) shows the shows 83.33% protection and delays the onset of time of convulsion. Group IV: Test control 1 (ethanol extract of plant shows the 63.66% protection and delays the onset of time of convulsion). Group V: Test control 2 ethanol extract of plant shows the 83.33% protection and delays the onset of time of convulsion

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Table 3: Effect of ethanol extract of Pandanus odoratissimus Linn in picrotoxin induced convulsion in mice


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MES induced convulsion in mice

Low and high dose of Ethanol extract of P. odoratissimus Linn (100 and 200 mg/kg, respectively) and marketed sample of P. odoratissimus Linn 0.5 ml for chronic study of MES induced convulsion model in mice is used. The above mentioned doses were administered daily once for a period of seven consecutive days. The above mentioned doses exhibited a significant anticonvulsant effect by delays the Latency of tonic extensor phase and tonic-clonic seizures. The lower and higher doses of Ethanol extract and dose of marketed sample offers a protective effect of 66.66% and 83.33% up to 1 h interval respectively. The standard drug diazepam (5 mg/kg) exhibited a significant anticonvulsant activity and offered 100% protection [Table 4] and [Figure 3].
Figure 3: Maximal electroshock test induced convulsion in mice. Group I: Control group (shows 100% mortality). Group II: #Diazepam (shows 100% protection against convulsion). It does not produce any convulsion. Group III: Test control (marketed sample) shows the shows 83.33% protection and delays the onset of time of convulsion. Group IV: Test control 1 (ethanol extract of plant shows the 63.66% protection and delays the onset of time of convulsion). Group V: Test control 2 ethanol extract of plant shows the shows 83.33% protection and delays the onset of time of convulsion

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The observations of the present study are presented in [Table 2], [Table 3], [Table 4]. Tests of significance were done by ANOVA followed by Dunnett's test. P < 0.05 were considered to be significant. The results are analyzed and discussed. [18],[19]
Table 4: Effect of ethanol extract of Pandanus odoratissimus Linn in maximum electroshock induced convulsion in mice


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   Discussion Top


The outcome of this study provides evidence that the ethanol extract of the leaf of P. odoratissimus possesses anticonvulsant activity. The ability of P. odoratissimus to delay the onset of convulsions and/or shorten the duration of convulsions was considered an indication of anticonvulsant activity. [27],[28],[32],[33] Judging from the data obtained, the plant extract exhibited anticonvulsant activity in the Pentylenetetrazole (PTZ) test just like diazepam which can be due to action on GABA system. [34],[35] The exact mechanism by which PTZ produces seizures is not well-understood. It, however, has been shown to be due to inhibition and/or attenuation of GABAergic neurotransmission. [36],[37]

Currently, available anticonvulsant drugs are able to efficiently control epileptic seizures in about 50% of the patients, another 25% may show improvement whereas the remainder does not benefit significantly. Furthermore, undesirable side-effects from the drugs used clinically often render treatment difficult so that a demand for new types of anticonvulsants exists. One of the approaches to search for new anti-epileptic drugs is the investigation of naturally occurring compounds, which may belong to new structural classes. [4],[5]

Various classes of phytoconstituents such as alkaloids, lipids, terpenes, triterpenoids, flavonoids and coumarins have been reported to possess anticonvulsant activity. Anticonvulsant activity reports on such classes of phytoconstituents have been depicted .

PIC is a GABAA-receptor antagonist. [38],[39] GABAergic ionotropic receptors can mediate both pre- and post-synaptic inhibition. Pre-synaptic inhibition mediated by GABA often leads to inhibition of neurotransmitter release from the excitatory arm. [30] PIC-induced seizures which are due to the decreased GABAA-receptor-mediated inhibition in turn promote the excitatory arm of the CNS mainly mediated by glutamate. [35],[36] The P. odoratissimus extract being effective in the PIC-induced seizure test points to a more specific action on GABA-mediated neurotransmission. [24],[25],[26],[29],[30],[31]

Strychnine-induced seizure model, it is known that strychnine a potent spinal cord convulsants, blocks glycine receptor selectively to induce excitatory response in the CNS. The P. odoratissimus does not offered a significant inhibition against SIC; if it might be interfere with glycine transmissions. The suppression of seizures by P. odoratissimus extract as compared with std. drug Diazepam might be indirectly enhancing glycine inhibitory mechanisms. [20],[21],[22],[23]

The extract showed no effect in the MES at all doses. This indicates that it is slightly effective in partial and generalized tonic seizures [40] and is able to prevent seizure spread. [41] It was also ineffective in SIC. SIC by antagonizing competitively the postsynaptic inhibitory effects of glycine. [42] The fact that P. odoratissimus extract produced no protective effects against SIC suggests it does not interact with the glycine-mediate inhibitory pathway. [43],[44]


   Conclusion Top


Therefore, the results obtained from the study suggest that Ethanol extract of leaves of P. odoratissimus Linn has possesses anticonvulsant property against the PIC and MES induced seizures via possibly through GABA-mediated inhibition and inhibition of glutamate mediated excitation via activation of potassium ion channels or non-specific mechanisms and the results verify its traditional use in epilepsy. Further phytochemical studies are in progress to isolate, characterize and identify the specific active compounds in this plant responsible for anti-convulsant activity.

However, extensive studies are needed to evaluate the precise mechanism (s), active principles and the safety profile of the plant as a medicinal remedy for convulsive disorders.

 
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    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]


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