Parkinson’s disease (PD) is a progressive neurodegenerative disorder, whose treatment with modern therapeutics leads to a plethora of side effects with prolonged usage. Therefore, the management of PD with complementary and alternative medicine is often pursued. In the Ayurveda system of alternative medicine, Yashtimadhu choorna, a Medhya Rasayana (nootropic), prepared from the dried roots of Glycyrrhiza glabra L. (licorice), is prescribed for the management of PD with a favorable outcome. We pursued to understand the neuroprotective effects of Yashtimadhu choorna against a rotenone-induced cellular model of PD using differentiated IMR-32 cells. Cotreatment with Yashtimadhu choorna extract rescued rotenone-induced apoptosis and hyperphosphorylation of ERK-1/2. Quantitative proteomic analysis of six peptide fractions from independent biological replicates acquired 1,561,169 mass spectra, which when searched resulted in 565,008 peptide-spectrum matches mapping to 30,554 unique peptides that belonged to 4864 human proteins. Proteins commonly identified in biological replicates and >4 PSMs were considered for further analysis, leading to a refined set of 3720 proteins. Rotenone treatment differentially altered 144 proteins (fold ≥1.25 or ≤0.8), involved in mitochondrial, endoplasmic reticulum, and autophagy functions. Cotreatment with Yashtimadhu choorna extract rescued 84 proteins from the effect of rotenone and an additional regulation of 4 proteins. Network analysis highlighted the interaction of proteins and pathways regulated by them, which can be targeted for neuroprotection. Validation of proteomics data highlighted that Yashtimadhu confers neuroprotection by preventing mitochondrial oxidative stress and apoptosis. This discovery will pave the way for understanding the molecular action of Ayurveda drugs and developing novel therapeutics for PD.

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