INTRODUCTION:

Central nervous system (CNS) disorders are of great concern in the present day world due to increasing stress and changing living conditions. CNS disorders are complex disease states that represent major challenge for modern medicine. Since epilepsy is one of the most prevalent CNS disorders and as a number of side effects are associated with the present antiepileptic drug treatments (Chimakurthy et al., 2008). Epilepsy is a common neurological disorder affecting an estimated 40-50 million people worldwide (Rudiger, 2002). Epilepsy has now become the most serious brain disorder, which accounts for about 1% of the world’s burden of diseases. Epilepsy is characterized by recurrence of seizures, the manifestation of paroxysmal and disordered neuronal discharges in the brain. Epilepsy can be caused by imbalance in the GABA nergic system. γ - aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian central nervous system (CNS) (Kahnberg et al., 2002).

Monotherapy is not effective in all cases of epilepsy. Still 20–30% patients with epilepsy remain refractory to monotherapy. At present, using rational medication (monotherapy or polytherapy), developing new antiepileptic drugs (AEDs) and neurosurgery are three chief strategies to deal with refractory epilepsy (Schmidt and Gram, 1995).

Although several drugs are available to treat epilepsy, the treatment of epilepsy is still far from adequate (McNamara, 1996). Many attempts have been made in the past to obtain anticonvulsant from plant origin and these efforts will continue till a satisfactory treatment becomes available (Sonavane et al., 2002). Numerous herbal medicines are recognized as active in the CNS disorder viz., epilepsy that do not respond well to conventional treatments (Phillipson, 2001; Carlini, 2003). Presently, scientists are keen to obtain drugs from plant origin due to their specific curative properties and relative low adverse effect. The Ayurvedic system of medicine has a quite sophistical classification of medicinal plants as per the dominant pharmacological/ therapeutic activity of mental functions (Vaidhya, 1997).

The plant Boswellia serrata Roxb., (Burseraceae), is a medium-sized tree of great economic value, distributed in the dry hilly forests of Rajasthan, Madhya Pradesh, Gujarat, Bihar, Assam and Orissa in India (Kirtikar and Basu, 1981). The bark of the tree yields a milky liquid that oozes out and congeals slowly into a gum resin (also known as ‘sallai-guggal’ or ‘olibanum’). Sallai-guggal is used in the treatment of rheumatoid arthritis (Trubestein et al., 1999) and also in various Ayurvedic preparations, such as Karpuradyaoka, Tisakada, Modaka etc. (Anonymous, 1978; Gupta et al., 1987). A literature survey revealed that its pharmacological effects are mainly attributed to the presence of pentacyclic and tetra cyclic triterpenoids known as boswellic acids (BAs) (Ammon, 2002). BAs were first isolated in 1932 (Winterstain et al., 1932). The BAs is a mixture of four major pentacyclic triterpene acid viz., β-boswellic acid, 3-acetyl β-boswellic acid, 11-keto β-boswellic acid and acetyl-11-keto-boswellic acid (BA). Amongst them, BA has been shown to be the most effective constituent (Gupta et al., 1997). The BAs are reported to be effective as a leukotriene inhibitor in neutrophiic granulocytes by a nonredox, noncompetitive inhibition of 5-lipoxygenase (Safahi et al., 1997; Safahi et al., 2000). Certain BAs have been described to inhibit elastase in leukocytes, to inhibits proliferation, induce apoptosis and to inhibits topoisomerases of leukoma and glioma cell lines, to inhibit lipopolysaccharide-mediated tumor nacrosis factor (TNFα) induction in monocytes by direct interaction with IkB kinase (Syrovets et al., 2000; Syrovets et al., 2005). In clinical trials, promising results were observed in patient with rheumatoid arthritis inflammatory bowel diseases when treated with Bas (Ammon, 2002; Gerhardt et al., 2001; Gupta et al., 2001).

Hence, the present study has been carried out to evaluate the BA for the anticonvulsant activity against chemical-induced convulsion models and effect of thiopental sodium induced sleeping time in experimental animals.

MATERIALS AND METHODS:

2.1 Drugs and chemicals

Diazepam (DZ) was purchased from Sigma (St. Louis, MO, USA). Thiopental sodium was purchased from the market. The standard BA was purchased from Yucca Enterprises, Mumbai. All other chemicals used were of the highest purity commercially available.

2.2 Plant material

The gum resin of the plant was collected from the local area surrounded by Saurashtra University campus, Rajkot, Gujarat, India, and authenticated by Prof P. J. Parmar, Botanical Survey of India, Jodhpur, Rajasthan (India). A herbarium was prepared (voucher specimen no. SU/DPS/Herb/29) and deposited at the Department of Pharmaceutical Sciences, Saurashtra University, Rajkot, India, for future reference.

2.3 Extraction and isolation of BA

The fraction containing BAs was prepared by extracting B. serrata gum resin (1 kg) successively with ethanol (95% v/v) in a percolator and evaporated under reduced pressure on a thin film evaporator at 40 ºC to obtain a thick brown residue (490 g). The total extract was stirred with 3% sodium hydroxide (NaOH) solution till it produced a uniform emulsion. The aqueous part was filtered and extracted with hexane: ethyl acetate (95:5) to remove the nonacidic part. The aqueous portion was then acidified with 1 N hydrochloric acid to precipitate the total organic acids. The filtered acids were washed with distilled water to remove final traces of hydrochloric acid. The crude mixture of acids was redissolved in 3% NaOH solution and whole process was repeated till precipitation was complete. The precipitates were dried in a vacuum oven at temperature below 50ºC to yield 280 g creamish powder of BAs (Singh et al., 2008).

2.4 Animals

Swiss albino mice (25-30 g) of either sex were obtained from the central animal house of the department and used for further study. The animals were housed at room temperature (25 ± 1 ºC) with 50-55% relative humidity and given standard laboratory feed (Pranav Agro Industries Ltd., India) and water ad libitum. The study was conducted after obtaining ethics committee clearance from the Institutional Animal Ethics Committee (Protocol approval no. VIP/IAEC/2011-12/29). For the present study, animals were randomized into five groups of six animals each and allowed to acclimatize for one week before the experiments.

2.5 Pentylenetetrazole-induced seizures (PTZ) in mice

The minimal i.p. dose of PTZ at which 99.9% of the animals showed HLTE (Swinyard, 1969) was determined by a dose-percent effect curve (Litchfield and Wilcoxon, 1949). The dose of PTZ (90 mg/kg) was given to five groups of animal each with six mice, pretreated i.p. with saline (10 ml/kg, as control), diazepam (4 mg/kg, as positive control) and BA (50, 100, 200 mg/kg). During 45 min test period, the animals were observed for various symptoms like ear and facial twitching, convulsive waves axially through the body, myoclonic body jerks, straub’s tail, generalized clonic convulsions, twitching and turning into one side, hind limb tonic extensor (HLTE) phase, stupor, and recovery or death. An animal was considered as convulsed, if it showed HLTE phase (Yemitan and Adeyemi, 2005; Quintans et al., 2008). The time of peak effect of diazepam (30 min after administration) was previously established (Pourgholami et al., 1999). The time for the BA to reach its maximum effect was determined as 30 min after i.p. injection. If no HLTE occurred during 30 min period of observation, the animals were considered protected and the ED50 and its associated 95% confidence limit were then determined (Litchfield and Wilcoxon, 1949).

Seizure intensity was evaluated using the following modified scale (Erakovic et al., 2001);

Stage 0: No response

Stage 1: Ear and facial twitching

Stage 2: Convulsive waves axially through the body

Stage 3: Myoclonic body jerks and straub tail

Stage 4: Generalized clonic convulsion, turn over into side position

Stage 5: Generalized convulsions with tonic extension episode and status epilepticus

Stage 6: Mortality

2.6 Thiopental sodium-induced sleeping time

The animals were divided in five group six mice each pretreated intraperitoneally (i.p.) with saline (10 ml/kg, as control), DZ (4 mg/kg, as positive control) and test doses of 50, 100 and 200 mg/kg of BA. After thirty minutes, five groups of mice received thiopental sodium (12 mg/kg) intraperitoneally. The time since the injection up to the loss of the righting reflex is recorded as sleeping latency and the time elapsed between the loss and voluntary recovery of the righting reflex is recorded as sleeping time (Wambebe, 1985; Rolland et al., 1991).

2.7 Statistical analysis

All the data are presented as mean ± S.E.M. The significance of difference in means between control and treated animals for different parameters was determined by using one-way analysis of variance (ANOVA) followed by multiple comparisons Dunnett’s test. A value of p<0.05 was considered statistically significant.

RESULTS and DISCUSSION:

Screening for anticonvulsant activity

PTZ- induced convulsion in mice

In PTZ-induced seizures, the administration of BA, in a dose of 200 mg/kg, 30 min. prior to the injection of PTZ, significantly (p<0.001) delayed the onset of HLTE (Table 1). DZ in a dose 4 mg/kg totally abolished the episodes of convulsions (p<0.001). There was less significant effect of the BA at dose of 100 mg/kg on onset of HLTE (p<0.01) as compared to control (Table 1). There was no significant effect of BA at dose of 50 mg/kg on onset of HLTE (Table 1).

Thiopental sodium induced sleeping time in mice

The absolute values of the sleeping latency (onset of sleep) and sleeping time are presented in Table 2. No alteration was observed on onset of sleeping at any doses of BA as compared to control. However, the duration of sleeping (Table 2) was significantly increased (p<0.001) in BA (200 mg/kg) as compared to control. Similarly, animals treated with DZ (4 mg/kg, i.p.), as expected, an increase in duration of sleeping (p<0.001) and did show significant effects onset of sleeping (Table 2) (p<0.001). There was no significant effect observed on BA in doses 50 and 100 mg/kg, on onset of sleeping as well as on duration of sleeping (Table 2).

CONCLUSION:

In PTZ-induced seizures which correlate with anti-absence activity (Delgado, 1998). The BA had little protection on the animal against anticonvulsant activity in this model (Table 1). In this model, there were no significant alterations in the latency of HLTE at lower doses, but higher dose of BA showed significant protection against HLTE as compared to control. The standard drug DZ at a dose of 4 mg/kg of body weight, provide 100% protection and showed complete absence of HLTE. The higher dose of BA showed 50% protection against convulsion. However, these effects were not dose-dependent. Drugs that are effective against petitmal seizures reduces T- type calcium currents, and these types of seizures can also be prevented by drugs that enhance GABAA-BZD receptor mediated neurotransmission, such as benzodiazepines and phenobarbitone (McDonald and Kelly, 1995). PTZ may be exerting its convulsant effect by inhibiting the activity of GABA at GABA receptors. GABA is the major inhibitory neurotransmitter which is implicated in epilepsy. The enhancement and inhibition of the neuro- transmission of GABA will attenuate and enhance convulsion respectively (Westmoreland et al., 1994). Antiepileptic drugs effective in the therapy of generalized seizures of (absence or myoclonic) petit-mal type such as phenobarbitone, valproate, ethosuximide and benzodiazepines exhibit dose-dependent suppression of various seizure pattern induced by PTZ (Loscher et al., 1991). In PTZ-induced convulsions, the BA had only increased the latency but not the incidence of seizures as compared to diazepam (Table 1). The activity observed in PTZ-induced studies may probably be due to possible interaction between BA and GABAergic neurotransmission.

The ability of the BA to exhibit activity against these two types of seizures suggests that it may act through different mechanisms to elicit its anticonvulsant effects, such as voltage-gated sodium, calcium, and potassium or GABAergic pathway. The CNS depressant activity of BA was confirmed by the decrease in the latency to sleep and its tendency to significantly increase the thiopental sodium-induced sleeping time (Table 2) which may be attributed to an inhibition of thiopental sodium metabolism or to an action in the regulation of sleep (Morais et al., 1998).

Table- 2. Effect of BA on Thiopental sodium-induced sleeping time

Group	Dose (mg/kg, i.p.)	Onset of sleep (min)	Duration of sleep (min)
Control	Vehicle	3.14 ± 0.11	8.32 ± 0.47
DZ	4	1.97 ± 0.20***	40.42 ± 1.86***
BA	50	3.17 ± 0.10	8.60 ± 0.59
BA	100	3.59 ± 0.18	9.892 ± 0.34
BA	200	2.53 ± 0.23	29.57 ± 1.56***

Values are expressed as mean ± SEM, n = 6, One-way Analysis of Variance (ANOVA) followed by multiple comparison Dunnett’s test, ***p<0.001 vs. Control.

The sleeping time was increased significantly at higher dose of BA, as compared to control. Animals treated with DZ (4 mg/kg, i.p.), as expected, prolong the sleeping time and reduces the latency of sleep. There was no significant effect on latency as well as sleeping time at lower doses of BA we have used two different animal model experiments that characteristically described two types of seizures activity. The electrically induced seizure signifies activity against generalized tonic-clonic and partial seizures. In MES-induced convulsion, the BA signiﬁcantly protected the animals against seizures, increased the onset and reduced the duration of seizures.

The majority of currently available antiepileptic drugs fall into one of two pharmacological classes, those that modulate neuronal voltage-gated sodium channels (e.g. carbamazepine, phenytoin, lamotrigine, and topiramate) and those that modulate inhibitory GABAergic neurotransmission (e.g. benzodiazepine, vigabatrin and tiagabine). While, small number of AEDs such as ethosuximide, gabapentin and possibly levetiracetam, may exert their effects via an interaction with voltage-operated calcium channels (Wickenden, 2002). The ability of the BA to exhibit activity against these two types of seizures suggests that it may act through different mechanisms to elicit its anticonvulsant effects, such as voltage-gated sodium, calcium, and potassium or GABAergic pathway.

The results of the study have demonstrated that BA possessed anticonvulsant activity on the animal model investigated and this provides a rationale for its use in traditional medicine for the management of epilepsy. Further studies are required to find out the exact mechanism of action of BA against the various animal models of convulsions.

ACKNOWLEDGEMENTS:

I am thankful to Dr. G. Vidya Sagar, Professor and Principal, Veerayatan Institute of Pharmacy for providing me infrastructure working facility for my project work and also to Dr. P. M. Patel, Professor and Principal, Kalol Institute of Pharmacy, for providing the knowledge about my work.

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Received on 28.06.2013 Modified on 06.07.2013

Research J. Pharm. and Tech. 6(9): September 2013; Page 1037-1041

Group	Dose (mg/kg, i.p.)	Onset of HLTE (min)	Convulsion (%)	Protection (%)
Control	Vehicle	3.59 ± 0.28	100.00	00.00
DZ	4	00.00 ± 0.00***	0.00	100.00
BA	50	4.25 ± 0.35	100.00	0.00
BA	100	5.52 ± 0.34**	83.33	33.33
BA	200	10.08 ± 0.50***	50.00	50.00