Indian Journal of Pharmacology Medknow Publications on behalf of Indian Pharmacological Society ISSN: 0253-7613 EISSN: 1998-3751 Vol. 37, Num. 2, 2005, pp. 120-125

Indian Journal of Pharmacology, Vol. 37, No. 2, March-April, 2005, pp. 120-125

Research Paper

Investigations of Sapindus trifoliatus in dopaminergic and serotonergic systems: Putative antimigraine mechanisms

Arulmozhi D.K., Veeranjaneyulu A., Bodhankar S.L., Arora S.K.

New Chemical Entity Research, Lupin Research Park, Village Nande, Taluk Mulshi, Pune-411 042

Correspondence Address:New Chemical Entity Research, Lupin Research Park, Village Nande, Taluk Mulshi, Pune-411 042. E-mail: addepalli@lupinpharma.com; add_bits@yahoo.com

Code Number: ph05022

ABSTRACT

INTRODUCTION

Drugs

Apomorphine, pargyline, haloperidol and l-5-HTP were obtained from Sigma Chemical Co (ST. Louis, USA). MK-801 was obtained from RBI (USA) and methysergide was procured from Tocris (Avonmouth, UK). Except haloperidol, which was dissolved in 0.01% lactic acid solution, all other drugs and chemicals were dissolved in normal saline. All the drugs were administered intraperitoneally.

Plant material and extraction procedure

Pharmacognostically identified dried pericarp of fruits of Sapindus trifoliatus Linn, family Sapindaceae were collected from the local market. Aqueous extract of ST was prepared as reported.[14] Briefly, 100 grams of the pericarp was soaked in 400 ml of distilled water for 16 h. The percolate was then decanted, centrifuged and filtered through Whatman (No.1) filter paper to obtain a clear extract (300 ml). The extraction process was repeated again with the same volume of distilled water. The percolates were pooled and lyophilized to give a brown colored powder (68% yield). Acid hydrolysis of the extract yielded only one glycone, which was identified as hederagenin. Therefore, estimation of the saponins present in the extract was calculated as hederagenin. The content of hederagenin in the extract was estimated by boiling it with 50% methanolic hydrochloric acid. The entire mixture was evaporated to dryness and reconstituted in methanol. HPLC estimation was carried out using Kromasil C-18 column (5 µm, 250 x 4.6 mm) with gradient elution (0.1% formic acid and acetonitrile in the ratio of 80: 20 v/v) and evaporative light-scattering detector. The concentration of hederagenin was found to be between 5.61-6.97 % by weight of the extract.[15]

Animals

Adult male Swiss albino mice (18-22 g) were obtained from the Research Animal Facility of Poona College of Pharmacy (PCP), Pune, India. On arrival, the animals were placed at random and allocated to treatment groups (10 animals per treatment) in polypropylene cages with paddy husk bedding. They were housed at a temperature of 24±2 ºC and relative humidity of 30-70% with a 12:12 light:dark cycle. All had free access to water filtered through Aquaguard ® and standard pelleted laboratory animal diet. The experimental procedures and protocols used in the present study were reviewed and approved by the Institutional Animal Care and Use Committee of PCP, Pune, India, and were in accordance with the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Forests and Environment, Government of India. Experiments were carried out between 1000 and 1700 h.

Apomorphine-induced climbing behavior

The method of Chung et al [16] was followed with minor modifications. Each mouse was placed in a cylindrical wire mesh cage (height 13 cm, diameter 14 cm and mesh size 3 mm) for 1 h prior to the experiments. Saline (10 ml/kg, i.p.) or ST (20 or 100 mg/kg, i.p.) or haloperidol (1 mg/kg, i.p.) was administered 30 min prior to apomorphine (2.5 mg/kg, i.p.). Climbing behavior was assessed at 5-min intervals up to 20 min, starting 10 min after apomorphine, using the following scoring system. 0-no paws on the cage, 1-two paws on the cage, 2-four paws on the cage. The score recorded for each animal was based on the position of the animal at the moment it was first observed. The total time spent on the cage was also recorded for each animal. An observer unaware of the specific treatments recorded the observations.

l-5-HTP-induced serotonin syndrome

The syndrome was measured after placing a mouse in a Perspex cage for a 30-min habituation period. The animal was then injected with pargyline (75 mg/kg, i.p.) in order to prevent the rapid degradation of l-5-HTP. Thirty min later, ST (20 or 100 mg/kg, i.p.) or methysergide (10 mg/kg, i.p.) was administered. After 30 min the animal received l-5-HTP (50 mg/kg, i.p.) and was returned to test cage. After 20 min, the serotonin syndrome was assessed every 10 min through 50 min. Five behavioral parameters, such as tremor, hind limb extension, forepaw treading, head weaving and head twitch were monitored using the following scoring system: 0-absent, 1-moderate, 2-marked.[16],[17] A trained observer unaware of the specific treatments recorded the findings.

MK-801-induced hyperactivity

Mouse locomotor activity was measured using a computerized animal activity meter (Opto Varimex Auto Track System, Columbus Instruments, OH, USA), made of clear perspex box (40 x 40 x 40 cm). An array of 15 infrared emitters (spaced at 2.65 cm intervals; beam wave length 940 nm) measured the animal′s activity along the single axis of motion. To minimize the variation among the individuals, mice were placed in pairs in the locomotor activity boxes for a 30 min period. After this time, the animals were injected with ST (20 or 100 mg/kg, i.p.) or haloperidol (0.1 mg/kg, i.p.) or vehicle and then returned to the test boxes. The recording was begun immediately. After a 30 min period, the animals were injected with MK-801 (0.3 mg/kg, i.p.) and the recording was continued immediately. Activity was measured for a further 90 min. The perspex boxes allowed for continuous visual monitoring of the animals in addition to the automated measurement of locomotor activity. The photocell counts that were measured every minute were grouped into 10 min time intervals.[16],[18]

Receptor radioligand binding studies

The receptor radioligand binding studies were carried out by NovaScreen Biosciences Corporation, USA. The studies were done as per the standard protocols[19],[20],[21] and the details of the receptor source ligands and reference compounds used are listed in [Table - 1].

Statistical analysis

Results are expressed as mean±SEM. Comparisons between the groups were made by Student′s ′t ′ test or analysis of variance (ANOVA) followed by Dunnetts′ post test as per suitability. A P value of < 0.05 was considered as significant.

RESULTS

In receptor radioligand-binding studies, ST exhibited an affinity towards various receptors and enzymes that are relevant targets in migraine [Table - 1] at an optimal concentration of 250 µg/ml.[21]

The antimigraine gold standard sumatriptan was originally identified as a selective agonist at 5-HT₁-like receptor mediating contraction of dog/rabbit saphenous vein. Sumatriptan was soon proven to be beneficial in the treatment of acute migraine headache, a finding that generated considerable scientific interest towards the identification of the selective serotonin receptor involved in the contraction of the cerebrovascular smooth muscle. While it was reported to act on the 5-HT₁-like receptor causing contraction of the dog/rabbit saphenous vein, it was pointed out that there were strong pharmacological similarities between the 5-HT_1B and/or 5-HT_1D receptors and 5-HT₁ receptor mediating cerebral vasoconstriction in the cat and human cerebral arteries. Indeed, now there is overwhelming evidence that the 5-HT_1B receptor mediates the contraction of vascular smooth muscles. In line with the above, ST has been studied for its potential affinities towards 5-HT_1B/1D receptors in rabbit saphenous vein.[22] However, ST failed to exhibit an affinity towards acute migraine targets viz. α-adrenoceptors and 5-HT_1B receptors in rabbit aorta and rabbit saphenous vein respectively.[22] Since ST has been used in the treatment of hemicrania in folklore medicine and the role of 5-HT_1B and α-adrenoceptors was ruled out in functional studies, the possible effects of ST through 5-HT_2A and dopamine D₂ receptor mediation were studied in the present investigation.

In the present investigation, ST exhibited pharmacological actions mediated through dopamine and serotonin receptors in the central nervous system. The current data is in conformity with the ligand binding studies, where ST (250 µg/ml) exhibited affinity towards dopamine D₂ (104.54%) and serotonin (5HT_2A) (47.53%) receptors.

Apomorphine-induced climbing response is reported to be mediated through the activation of both D₁ and D₂ receptors,[23] and D₁ and D₂ antagonists were proved to be efficacious in this model.[24]

The prophylactic antimigraine methysergide, a serotonin (5HT₂) antagonist, reportedly inhibited the components of l-5-HTP-induced behavioral syndromes, especially the head twitch response.[25]

The locomotor stimulant effects of MK-801 (a non-competitive NMDA channel blocker) have been correlated to increased dopamine and serotonin release in the brain.[26],[27] Dopamine (5HT₂) and serotonin antagonists are reported to antagonize MK-801-induced hyperactivity in mice.[18] Recent pharmacological data have demonstrated that MK-801 increases 5-HT turnover in several brain regions including the cortex, hippocampus, nucleus accumbens, amygdala and striatum. In addition to direct glutamatergic-serotonergic interactions, dopaminergic-serotonergic interactions might be involved in the alteration of serotonin turnover produced by NMDA antagonists.[28] The serotonergic system modulated through 5-HT_2A receptors could play a permissive role in the regulation of forebrain dopaminergic systems via glutamate.[29],[30] The ability of MK-801 to increase dopamine and serotonin release in brain favors the notion that increased monoaminergic neurotransmission underlies the locomotor stimulant effect.[27]

ST, a phytomedicine used for the treatment of hemicrania, exhibited dopamine D₂ and serotonin (5HT₂) antagonism by inhibiting the behavioral effects induced by dopamine and serotonin agonists in the aforementioned animal models. ST exhibited less affinity towards 5-HT_2A receptors than dopamine D₂receptors in the ligand binding studies, which may be related to the higher dose required for the significant inhibition of 5-HT-induced syndromes [Figure - 3].

Clinical and pharmacological evidence suggests that the involvement of dopamine in migraine and most prodromal and accompanying symptoms could be related to dopaminergic activation. Furthermore, migraine patients show a higher incidence of dopaminergic symptoms following acute dopamine agonist administration, when compared with normal volunteers.[31] Migraine associated with nausea and vomiting is often controlled with dopamine receptor antagonist metoclopromide.[32]

A growing body of biological, pharmacological and genetic evidence suggests the role of dopamine in the pathophysiology of certain types of migraine. Most migraine symptoms can be induced by dopaminergic stimulation. Moreover, there is dopamine receptor hypersensitivity in migraineurs, as demonstrated by the induction of yawning, nausea, vomiting, hypotension and other symptoms of migraine attack by dopaminergic agonists at doses that do not affect non-migraineurs. Conversely, dopamine receptor antagonists are effective therapeutic agents in migraine.[7] Recent genetic data suggest that molecular variations within dopamine receptor genes play a modifying role in the pathophysiology of migraine with aura.[33],[34] In a recent study, dopamine D2 antagonist prochlorperazine showed efficacy in hyperalgesic mice, claimed to be an in vivo model for migraine.[35]

It has been well established that serotonin is implicated in the pathophysiology of migraine and several drugs with potent serotonin (5HT₂) receptor blocking activity (methysergide, pizotifen, cyproheptadine and mianserin) have been recognized as being clinically effective in migraine prophylaxis.[36] The 5-HT_2A receptor mediated actions of serotonin relevant to migraine include increased cranial capillary permeability and platelet aggregation.[36] It is also reported that activation of 5-HT_2A  receptor leads to an enhancement of NO production in the trigeminovascular pathway. NO may trigger migraine attack by inducing cerebral vasodilation and sensitizing the perivascular nociceptors and central nociceptive neurons in the trigeminovascular systems.[37] An up-regulation of 5-HT_2A  receptor has been demonstrated to be correlated with the frequency of migraine attacks. Interestingly, at a concentration of 250 µg/ml, ST has an 84% inhibition of NOS (constitutive neuronal) in radioligand binding studies [Table - 1]. Nitroglycerin-induced hyperalgesia is considered to be a model of migraine headaches. It is evident from the literature that nitroglycerin produces dose-dependent headache in normal volunteers and migraine sufferers.[38] Furthermore, we have recently reported the antinociceptive activity of ST in nitroglycerin-induced hyperalgesic rats.[14] In a double-blind study, selective serotonin (5HT₂) receptor antagonist ritanserin was highly effective in reducing Pain Total Index and analgesic consumption in chronic headache.[39]

In view of the modulatory role of ST on dopamine D₂ and serotonin (5HT₂) receptors, there is a strong possibility of the involvement of these receptors in its antimigraine activity. The present data adds value to the reported ethnomedical usage of ST in the treatment of hemicrania.

ACKNOWLEDGEMENTS

REFERENCES

Copyright 2005 - Indian Journal of Pharmacology

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