Neuroprotective effects of an active constituent derived from Indigofera tinctoria extract in experimental models of Parkinsons disease.

Abstract

Introduction: As part of our drug discovery program for potential molecules in treating Parkinson's disease (PD), we have screened and isolated an active constituent from Indigenous herb Indigofera tinctoria Linn (I. tinctoria, Fabaceae) in experimental models of PD. I. tinctoria, is an annual herb found throughout India and other parts of Asia. The herb is widely used for several years in the traditional Indian and Chinese system of Medicine for the treatment of epilepsy, nervous disorders, bronchitis, liver ailments, sores, ulcers and hemorrhoids. Methods: Bio-assay guided extraction, fractionation, separation and identification of the active constituent from I. tinctoria extract was done through thin layer chromatography (TLC) and High Performance Liquid Chromatography (HPLC) and NMR techniques. To evaluate the in vitro protective activities the butanol sub-fraction, SF-6 was assessed using human neuroblastoma SH-SY5Y cells against α-synuclein-, 6-hydroxydopamine (6-OHDA)-, and H2O2- induced cytotoxicity using WST-1 cell viability assay. Further, the free radical scavenging effects of SF-6 was also evaluated using electron spin resonance (ESR) spectroscopy. The single compound methylparaben (MP) isolated from sub-fraction SF-6 was evaluated for reactive oxygen species (ROS) generation inhibition in 6-OHDA–induced cytotoxicity using DCF-DA in SH-SY5Y cells. MPs effect on lipid peroxidation assay was examined using the MDA analysis kits in 6-OHDA-induced mouse brain samples. In vivo studies were evaluated against 6-OHDA-lesioned neuronal damage by stereotaxically injecting 6-OHDA unilaterally into the substantia nigra. Agents were administered intraperitoneally 30 min before and 90 min after lesion induction. Animals received a further daily injection of agents intraperitoneally for 14 days. Two weeks after the 6-OHDA injection, contralateral rotational asymmetry was observed by apomorphine challenge. Further, to investigate the behavioral deficits induced by intra-nigral administration of 6-OHDA in mouse, rotarod test was also performed in the 6-OHDA-lesioned mice. Tyrosine hydroxylase-positive (TH+) cells in the substantia nigra of 6-OHDA-lesioned mice brain sections was performed by Immunohistochemical analysis using ABC kits. Results: The butanol sub-fraction, SF-6 attenuated the α-synuclein- induced cytotoxicity in SH-SY5Y cells and scavenged directly hydroxyl free radicals as estimated using the ESR spectroscopy. The single compound MP isolated from butanol sub-fraction (SF-6) showed significant and potent neuroprotective effects in both in vitro and in vivo evaluations. MP at nanomolar concentrations inhibited 6-OHDA- and H2O2-induced cytotoxicity and reduced the ROS generation in SH-SY5Y cells in a dose dependent fashion. Further MP attenuated the lipid peroxidation induced by 6-OHDA toxicity in mouse brain tissues. In in vivo studies, MP attenuated the behavioral deficits provoked by 6-OHDA injection and attenuated the contralateral rotational asymmetry in apomorphine challenged mouse in a dose dependent fashion. MP was found to be more potent when compared with known compound deprenyl. Further immunohistochemical analysis of brain tissues in MP treated subjects protected the dopaminergic neurons as evident by significantly higher number of surviving tyrosine hydroxylase-positive (TH+) cells in the substantia nigra of 6-OHDA-lesioned mice. Conclusions: Considering the results obtained our in vitro and in vivo findings suggest the possibility of MP to show neuroprotection in experimental models of PD via the antioxidant properties and therefore can further be developed as potential agent for treating neurodegenerative processes seen in PD. Keywords: Indigofera tinctoria, methylparaben, 6-hydroxydopamine

reactive oxygen species

Parkinsons disease, substantia nigra, SH-SY5Y cells

neurodegeneration

oxidative stress.