Pharmacognostical standardization and evaluation of anticoagulant potential of wild Himalayan pear : Pyrus pashia Buch. Ham ex D.Don

Abstract

Introduction For centuries people have used plants for healing. Natural products have been used with varying success to cure and prevent diseases throughout history, and continue to provide mankind with new remedies. People often resort to traditional and other forms of complementary and alternative medicines for chronic conditions which do not respond well to conventional or modern drug treatments. Among these are neurological disorders such as anxiety, depression, epilepsy and pain. The World Health Organization (WHO) estimates that about 75 % of the world population primarily those of developing countries depend on traditional remedies (mainly herbs) for the healthcare of its people. Considering the great reliance on traditional medicinal plants for treatment of diseases and the potential for drug discovery, it becomes relevant to search for potent, effective and relatively safe plant medicines. Epilepsy is a chronic neurological condition characterized by recurrent, unprovoked, and sudden neuronal discharge of impulses. Several diverse mechanisms have been suggested in the development of epilepsy that includes abnormal ionic conductance, alteration in neuronal membrane functions, imbalance in inhibitory and excitatory neurotransmission or a combination of these. Several antiepileptic drugs (AEDs) are available which act on miscellaneous molecular targets to suppress the abnormal excitability of neurons, either by reducing the seizure discharges, or by preventing the spread of abnormal excitation. However, around 30% of epileptic patients still have ‘intractable seizures’ that means they are unresponsive to these agents. The use of available AEDs is linked with variety of side effects, and often results in augmentation of the associated comorbid conditions, particularly cognitive impairments. Unfortunately, many times these conditions remain under-recognized and therefore left untreated in the patients. Prolonged and extensive use of AEDs impart considerable amount of negative impact on lives of the patients, that includes increased suicidal risk, poor tolerability, more predisposition to psychiatric side effects, and sometimes development of pharmacoresistant epilepsy. Hence, a lot of research is being carried out in search of safe and effective agents that can also reduce the risk of epileptic comorbidities. Natural products have been found to be a better alternative for management of neurobehavioural epileptic comorbidities due to their multi-targeted nature. Natural products as one of the complementary and alternative medicine modalities offer potential opportunities to discover lead compounds for novel drug development. Though several herbs and their active ingredients have been studied in laboratory and clinical settings, only a few have been investigated Abstract 2 for their molecular mechanisms of action. Thus, the discovery of a new anticonvulsant drug with better efficacy and improved safety profile is of fundamental importance. Pyrus pashia (Hamilton ex D. Don; Family: Rosaceae) is a deciduous plant with small and ovate shaped toothed leaves, attractive white flowers, and small pear-like fruits. Native to Himalayan region of Northern India fruits of this plant are used traditionally to treat convulsions and epilepsy. Moreover its fruits are persuasive source of polyphenolics. Earlier research have established that these polyphenols are implicated in treatment of epilepsy. Polyphenols can interplay with variety of cellular receptors or proteins that are involved in signaling cascades, resulting in physiological responses or gene expression which leads to neuroprotection. Thus major objectives of present study are:  To establish the pharmacognostical and phytochemical standardization of Pyrus pashia fruits in order to ensure its identity.  The study is an attempt to scientifically evaluate the traditional claims of fruits of Pyrus pashia in epilepsy.  Isolation of its major bioactive component and establishing its mechanism of action. Material and Methods: Pharmacognostical standardization for the fruits of Pyrus pashia was done as per WHO guidelines followed by qualitative and quantitative evaluation of various secondary metabolites present in the extract. Furthermore, polyphenolic fingerprinting of ethanolic extract of Pyrus pashia (EPP) was done, to identify major polyphenolics present in EPP. Chrysin was then isolated from EPP since it was the major polyphenol in extract. The anticonvulsant activity in terms of duration of onset of hind limb tonic extension and duration of convulsion of standardized EPP and chrysin was evaluated against maximal electroshock (MES) and pentylenetetrazole (PTZ) model of experimental epilepsy. Moreover, the neurotoxic profile of the extract and chrysin was assessed in terms of duration of movement and running in photoactometer and rotarod apparatus. Furthermore in order to investigate the mechanism of action EPP and chrysin the latency for development of convulsions and mortality rate was recorded in different experimental mouse models of epilepsy like strychnine induced convulsion, picrotoxin induced convulsion, isoniazid induced convulsion, 4-Aminopyridine induced convulsion, NMDA induced convulsion, further the effect of extract and chrysin on GABAA receptors was ascertained with the help of benzodiazepine antagonist, flumazenil. Chronic studies for the EPP and chrysin were done with pentylenetetrazole kindling model. Seizure severity was scored and cognitive function was tested Abstract 3 in the Morris water maze. Neuronal apoptosis was assessed by propidium iodide staining. Effect of EPP and chrysin on expression of apoptotic markers (cytochrome c, caspase 3, caspase 9) and neuroplasticity markers (BDNF) and gephyrin in hippocampus of epileptic mice was done using western blot analysis. Results: The study established quality control parameters for fruits of Pyrus pashia. The results from qualitative and quantitative estimation showed the extract of Pyrus pashia was persuasive source of polyphenols. Polyphenolic fingerprinting of EPP revealed that chrysin was the major polyphenol in the extract. EPP 200 and 400 mg/kg p.o. exhibited potent anticonvulsant activity against PTZ and MES models of convulsion, moreover chrysin 5 and 10 mg/kg p.o. showed significant protection against PTZ induced model. In addition, EPP and chrysin did not exhibit sedative like behavior in experimental rodents suggesting its safe profile. Anticonvulsant action of EPP and chrysin can again be correlated with the down regulation of apoptotic biomarkers and upregulation of BDNF and gephyrin levels in the hippocampus of experimental animals.Though EPP and chrysin (200 and 5 mg/kg; p.o.) exhibited similar therapeutic profile to that of diazepam (DZP), DZP-1 mg/kg; i.p. in the PTZ-induced cognitive and behavioural impairments, EPP and chrysin showed significant decrease in escape latency, these finding suggest that extract improved the impaired spatial memory of kindled animals. Moreover, both extract and its major bioactive component showed almost equal efficacy in terms of reduction in seizure severity, in kindling model of epilepsy indicating the fact that anticonvulsant activity of the EPP may be due to chrysin. EPP 200 and chrysin 5 were effective against strychnine, picrotoxin, isoniazid model of epilepsy. However treatment with EPP and chrysin failed to protect the mice from NMDA and 4-Amino pyridine induced convulsions. Above findings suggest that EPP and chrysin possess significant anticonvulsant activity and the outcomes further confirmed the involvement of GABAergic mechanism behind the anticonvulsant activity of EPP. Conclusion: Our study established the quality control parameters of Pyrus pashia fruit. Moreover further observations emphasize the fact that EPP could be considered as a potential and alternative therapeutic option in the management of epilepsy.