**1. Introduction**

Due to the low efficiency and toxicity of current medications for Parkinson's disease treatment, interest is growing in phytochemicals as a potential treatment option. Phytochemicals with neuroprotective activity target various pathways due to their antioxidant, anti-inflammatory, and antiapoptotic properties [1]. *Mucuna pruriens* Linn. is a leguminous plant growing spontaneously in tropical and subtropical areas worldwide. The seeds have traditionally been used in India as a nerve tonic, and a male virility enhancement [2]. In

**Citation:** Kamkaen, N.; Chittasupho, C.; Vorarat, S.; Tadtong, S.; Phrompittayarat, W.; Okonogi, S.; Kwankhao, P. *Mucuna pruriens* Seed Aqueous Extract Improved Neuroprotective and Acetylcholinesterase Inhibitory Effects Compared with Synthetic L-Dopa. *Molecules* **2022**, *27*, 3131. https://doi.org/10.3390/ molecules27103131

Academic Editors: Nour Eddine Es-Safi and Celestino Santos-Buelga

Received: 15 April 2022 Accepted: 11 May 2022 Published: 13 May 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

addition, the pods have anthelmintic activity, and the seeds have anti-inflammatory activity. Powdered seeds possess anti-parkinsonism properties, possibly due to the existence of L-dopa, which is a precursor of the neurotransmitter dopamine. The dopamine content in brain tissue is reduced when the conversion of tyrosine to L-dopa is blocked. L-Dopa can cross the blood–brain barrier and undergoes conversion to dopamine, restoring neurotransmission [3]. Particularly, the hydro-alcoholic extract of *M. pruriens* seeds gave high yields of L-dopa, using ascorbic acid as a protector [4]. Surprisingly, n-propanol extract of *M. pruriens* seeds that contained a small amount of L-dopa, yielded the highest reaction for the growth and survival of dopaminergic culture neurons [2]. The significant neuroprotective activity of n-propanol extracts suggested that a whole extract could be potential for the treatment of Parkinson's disease [5].

Parkinson's disease is characterized by signs of major oxidative stress and mitochondrial damage in the pars compacta of the substantia nigra [6]. The previous study suggested that reactive oxygen species (ROS) played an important role in age-related neurodegenerative changes including Parkinson's disease [7]. Interestingly, the ethyl acetate and methanolic extract of the whole *M. pruriens* plant exhibited high antioxidant and free radical scavenging activities [2]. These in vitro assays indicated that the whole plant extract contained large amounts of phenolic compounds, which may be useful in preventing various oxidative stresses. Furthermore, it has been reported that methanolic extracts of *M. pruriens var.utilis* leaves have numerous biochemical and physiological activities, and contain pharmaceutically valuable compounds [8]. However, the in vitro neuroprotective activity of the *M. pruriens* seed aqueous extract obtained from the traditional extraction method has never been reported.

Although the first-line therapy for Parkinson's disease is L-dopa, long-term L-dopa use results in the development of significant clinical complications. L-dopa-induced dyskinesia (LID) and abnormal involuntary movements (AIMs) normally occur in a vast majority of Parkinson's disease patients with repeated administration of L-dopa [9]. AIMs occurring at the head, trunk, and extremities affect the function of daily living, and patients can become debilitated. These side effects are common with 30% incidence after 2 years of L-dopa administration. The incidence increased up to 40% and 90% for 5- and 10-year-treatment, respectively [10]. Parkinson's patients who early started high doses of levodopa are known to have risk factors for LID [11]. At present, few therapeutic options are available for the treatment of LIDs. Several strategies have been proposed to reduce these side effects including postponing the initiation of L-dopa dosing, adjusting the dose of L-dopa, and combining it with other drugs [12,13]. However, these strategies resulted in a decrease in the efficacy of L-dopa in the treatment of Parkinson's disease.

The seed of *M. pruriens* has been widely investigated for its pharmacological properties including Parkinson's disease because it contains significant amounts of L-dopa. The advantages of natural L-dopa in *M. pruriens* seed extract over the synthetic forms have been reported. The natural L-dopa is less toxic [14]. It has a shorter onset of action, but it has a longer therapeutic effect, which could delay the need for combination therapy [14–16]. In this study, we quantified the amount of L-dopa, total phenolic content, and total flavonoid contents in freeze-dried *M. pruriens* seed extract obtained from an aqueous extraction method. The cytotoxicity and neuroprotective activities of the freeze-dried extract were investigated in neuronal cells using two models. The effects of L-dopa and *M. pruriens* on acetylcholinesterase enzyme activity were studied.
