**4. Discussion**

Parkinson's disease is a progressive neurodegenerative disorder resulting from the damaged dopaminergic neurons in the substantia nigra. The disease recently has no proven neuroprotective or neurorestorative therapy [27]. However, protecting neurons from premature cell death might be a promising alternative to managing this disease. Research directions include the investigation into animal models of the disease and the potential usefulness of gene therapy, stem cell transplant, neuroprotective agents, and herbals drugs.

The present study investigated the neuroprotective activities in P19-derived neurons using two models including the serum deprivation method and co-administration of hydrogen peroxide assay. Remarkably, the significant neuroprotective activities of standardized aqueous extracts of *M. pruriens* seed (L-dopa 7.05 ± 0.02 mg/100 g) were observed. The total phenolic compounds and total flavonoid content in *M. pruriens* seed aqueous extract were 106 GAE mg/g crude extract and 84 QE mg/g crude extract, respectively. Shin et al. have reported that L-dopa and pramipexol (a dopamine agonist) had comparable neuroprotective properties in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated Parkinson's disease animal models through modulation of cell survival and apoptotic pathway [28]. The total phenolic compounds have been shown to exert neuroprotective effects against hydrogen peroxide-induced oxidative damage by blocking reactive oxygen species production and improving mitochondrial function [29]. Flavonoids have demonstrated neuroprotective effect via the inhibition of cholinesterase enzymes including acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and β-secretase (BACE1) [30]. Therefore, our findings suggest that phenolic compounds and flavonoids in *M. pruriens* seed extract might play an important role in the neuroprotective effect against oxidative damage.

Regarding the serum deprivation method, the results demonstrated significant cell viability in 10 ng/mL *M. pruriens* extract (28.73 ± 1.90%) compared with the toxic condition (8.37 ± 0.73%). In addition, the results of the co-administration of hydrogen peroxide assay showed significant cell viability in 10 ng/mL *M. pruriens* extract (25.01 ± 2.66%) compared with the toxic condition (11.61 ± 0.50%). Our results agree with those of some previous studies. The evaluation of the anti-neuroinflammatory effects of *M. pruriens* extract was performed in murine microglia BV-2 cells. [16] *M. pruriens* extract was evaluated for neuroprotective effects in human neuroblastoma SH-SY5Y cells. The study showed that *M. pruriens* extract (at 1 μg/mL and 10 ng/mL) significantly reduced 6-hydroxydopamineinduced cytotoxicity and increased cell viability by 73.1 and 75.1%, respectively. The examination of ethanolic extract of *M. pruriens* on the level of nitric oxide in paraquat-induced Parkinson's disease mouse model and its subsequent contribution to lipid peroxidation results demonstrated that *M. pruriens* protects the dopaminergic neurons from the NO injury in substantia nigra [31]. The investigation of *M. pruriens* ethanolic seed extract in the Parkinsonian mouse model was performed [32]. A significant reduction in the activity of tyrosine hydroxylase positive neurons was observed in the substantia nigra region of the brain, after treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

Although the gold standard treatment of Parkinson's disease is L-dopa dopamine precursor, long-term L-dopa administration resulted in the development of serious side effects, including dyskinesia and the development of fluctuations in motor response. L-dopa-induced dyskinesia in Parkinson's disease patients significantly contributes to altered cholinergic signaling. Importantly, many studies showed that cholinergic receptor drugs including nicotine resulted in significant declines in L-dopa-induced dyskinesia [33–35]. Keeping the dose of L-dopa below 400 mg per day in early Parkinson's disease has been shown to reduce the risk of dyskinesia induction [36]. In this study, we showed that *M. pruriens* extract containing 7% natural L-dopa decreased the acetylcholine esterase stimulation compared with control. In contrast, the synthetic L-dopa significantly stimulated acetylcholinesterase activity. The results suggested that *M. pruriens* seed aqueous extract was probably able to modulate the cholinergic system and reduce L-dopa-induced dyskinesia and movement disorders resulting from L-dopa treatment. The present study demonstrated the neuroprotective activity of *M. pruriens* seed extract which may imply the therapeutic effects in various nervous system disorders including Parkinson's disease. Different mechanisms could underline this protection. Since *M. pruriens* seed extract showed neuroprotective activity against hydrogen peroxide-induced neurotoxicity, its proposed antioxidative therapeutics can be used for the protection of neuron injury induced by oxidative stress [37]. Serum deprivation-induced cell death is recognized as one of the standard models for the study of neurotoxicity. cGMP/PKG, PI13/Akt, and Bcl-2/Bax pathways have been shown to involve in the serum-deprivationinduced toxicity [38]. *M. pruriens* seed extract presented the potential of neuroprotective activity and thus may have a positive impact on aging and neurodegenerative diseases to retard the accelerated rate of neuronal degeneration.

*M. pruriens* seed extract contained substances including 5-hydroxytryptamine, alanine, arachidic acid, arginine, aspartic acid, behenic acid, beta-carboline, beta-sitosterol, bufotenine, choline, cis-12,13-epoxyoctadec-trans-9-cis-acid, cis-12,13-epoxyoctadec-trans-9-enoic-acid, cystine, gallic-acid, glutamic acid, glutathione, glycine, histidine, L-dopa, lecithin, leucine, linoleic acid, n, n-dimethyltryptamine, n, n-dimethyltryptamine-n-oxide, nicotine, phenyalanine, phosphorus, proline, protein, prurienidine, prurienine, saponins, serine, serotonin, threonine, tryptamine, tyrosine, valine, and vernolic acid, according to Dr. Duke's Phytochemical and Ethnobotanical Databases at Phytochemical Database, USDA-ARS-NGRL, Beltsville Agricultural Research Center [39]. Some of these compounds were AChE inhibitors, for example, physostigmine, which can enhance central levels of synaptic choline [40]. The aromatic amino acids such as tyrosine, phenylalanine, and tryptophan were identified as AChE inhibitors [41]. Pan et al. reported that flavonoid glycoside can inhibit AChE leading to a significant improvement in dyskinesia recovery rate in zebrafish [11]. Cognitive impairment and dementia are common complications of Parkinson's disease. Patients with Parkinson's disease with dementia often have significant cholinergic defects, which may be treated with cholinesterase inhibitors [42]. Several reports revealed that the use of an acetylcholinesterase inhibitor might improve cognitive function and reduce the risk of falls in patients with Parkinson's disease [43]. Therefore, *M. pruriens* seed extract with acetylcholinesterase inhibitory activity may be considered a prospective natural product for the treatment of Parkinson's disease, which may improve cognitive and motor functions of patients. This study reported the neuroprotective effect and anticholinesterase inhibition of *M. pruriens* seed aqueous extract, which contained large amounts of L-dopa (7%) along with other bioactive compounds including phenolic compounds and flavonoids. *M. pruriens* seed extract obtained in this study suggested more effectiveness and less toxicity than synthetic L-dopa for the treatment of Parkinson's disease. Our results were supported by Katzenschlager et al. showing the rapid onset of action and a long time without a concomitant increase in dyskinesias on *M. pruriens* seed powder formulation compared with L-dopa preparations in the long-term management of Parkinson's disease [14]. However, additional research is necessary to determine the agents responsible for other in vivo activities as well as the molecular mechanisms involved in their effects.
