INNOVATIVE ANALYTICAL METHODS FOR THE EVALUATION OF L-DOPA LEVEL IN TYPICAL LEGUMES OF BASILICATA TO ASCERTAIN THEIR POTENTIAL USE AS ADJUVANTS IN THE TREATMENT OF PARKINSON'S DISEASE

L-Dopa is a dopamine precursor widely used as the most effective drug for the symptomatic treatment of Parkinson's disease. Due to the neurotoxicity and side effects of synthetic L-Dopa, natural sources of this bioactive compound could be used as adjuvants to reduce unpleasant effects, particularly the 'on-off' motor fluctuations typical of severe Parkinson's disease. Natural L-Dopa offers advantages over its synthetic counterpart, including availability in its pure enantiomeric form and cost effectiveness. Some Fabaceae plants naturally contain significant amounts of L-Dopa. However, this bioactive compound is unstable in aqueous solutions and naturally degrades over time. Therefore, appropriate extraction and analytical methods are required for the unambiguous determination and quantification of L-Dopa in plant matrices. This research project is focused on the development of innovative analytical methods for the evaluation of L-Dopa levels in typical legumes grown in Basilicata (Vicia faba L. and Phaseolus Vulgaris L.). In the first part of the project an LC-UV method was optimized, validated according to the EURACHEM guidelines and tested to evaluate L-dopa levels in Vicia faba L. seeds stored under different conditions (fresh, sun-dried, freeze-dried, frozen, commercial long-life frozen). The extraction of the analyte from broad beans was carried out using an ultrasound-assisted extraction protocol (UAE), suitably optimized. A strongly acidic aqueous solution, consisting of HCl 0.1 M, proved to be the best extraction solvent and allowed to assure an appreciable stability of L-dopa standard solutions over 3 months. The optimal chromatographic conditions were achieved by using a Discovery C18 column (250 × 4.6 mm, 5 μm particle size) as a stationary phase and a mobile phase composed of 99% formic acid 0.2% v/v containing 1% methanol, under isocratic conditions. Sun drying was the best storage treatment for Vicia faba L. beans, capable of preserving the L-Dopa levels present in fresh samples (1.21 ± 0.17 mg/g dw). The validated LC-UV method proved to be a robust and efficient tool for analyzing plants with moderate levels of L-Dopa, such as Vicia faba seeds, but was not suitable for those with low levels, like Phaseolus vulgaris L. seeds. For this latter matrix, a method based on liquid chromatography coupled with tandem electrospray mass spectrometry (LC-ESI/MS/MS) was validated and applied to determine L-dopa content in four ecotypes of Fagioli di Sarconi beans marked with the European label PGI (Protected Geographical Indication). The selectivity was ensured by the specific fragmentation of the analyte. The LC–ESI/MS/MS method was validated according to the ICH (International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use) guidelines. Fresh and dried beans, as well as pods, were analyzed showing an L-dopa content ranging from 0.020 ± 0.005 to 2.34 ± 0.05 μg/g dry weight. In recent years, the by-products of Vicia faba L. have received renewed interest as a sustainable source of bioactive compounds. In this context, aqueous extracts from the pod valves of broad beans, organically grown by a local producer in San Chirico Raparo, Potenza, Italy, were characterized to assess their potential as adjuvants in the treatment of severe Parkinson’s disease. The L-Dopa content, quantified using LC-UV, was found to be significantly higher in freeze-dried Vicia faba L. pod valves compared to the seeds (28.65 mg/g dry weight vs. 0.76 mg/g dry weight). Notably, the metabolites vicine and convicine, which cause hemolytic crises in individuals with favism, were not detected in the pods. Using LC-ESI/LTQ-Orbitrap/MS², the major polyphenolic compounds in the aqueous extracts of the pods were identified, including quercetin and catechin equivalents, which may provide neuroprotective effects in Parkinson’s disease. Further analysis using ESI(±)-FT-ICR MS enabled the construction of 2D van Krevelen plots, a tool to obtain molecular formula maps, revealing the presence of polyphenolic compounds and carbohydrates as the most significant classes. The neuroprotective activity of the aqueous extracts of Vicia faba L. pod valves was demonstrated in vitro against the SH-SY5Y human neuroblastoma cell line, showing higher efficacy than synthetic L-Dopa even at concentrations up to 100 μg/mL, likely due to the synergistic effect of the antioxidant compounds that prevent the oxidative stress induced by the disease. To enhance the stability and antioxidant properties of the extracts, naturally acidic solutions from Punica granatum L. (5% w/v), Ribes rubrum L. (2% w/v), and Phyllanthus emblica L. (2% w/v) were used as extraction solvents. These solutions also resulted in lower protein content compared to pod valves extracts in ultrapure water, as evidenced by a comprehensive proteomic analysis using nLC-MS/MS (nanoflow liquid chromatography coupled with high-resolution tandem mass spectrometry) in Data-Dependent Acquisition (DDA) mode. Gene ontology (GO) analysis by biological process and semiquantitative profiling of major components revealed the presence of proteins such as chitinase in Vicia faba L. and Punica granatum L., which play important roles in plant defense and have been shown to inhibit fungal growth. This suggests that using naturally acidic juices for extraction could offer a "green" alternative to water, with the added benefit of reducing competition between L-Dopa and free amino acids during absorption in the brain. In the perspective of utilizing these natural extracts for the development of nutritional supplements to stabilize L-Dopa plasma levels in advanced Parkinson’s disease, a real-time monitoring device for L-Dopa levels would be advantageous. This would enable personalized dosing, optimizing therapeutic efficacy and minimizing side effects. An innovative amperometric biosensor based on tyrosinase co-crosslinked onto a graphene oxide layer, produced by electrodeposition, was optimized and validated for this purpose. Careful optimization of the enzymatic immobilization procedure ensured improved long-term stability and allowed the biosensor fabrication to be reduced and simplified. The effectiveness of the immobilization protocol, combined with the improved performance of the electrodeposited graphene oxide, ensured high sensitivity, a wide linear range and a detection limit of 0.84 M, suitable for the monitoring of L-Dopa within its therapeutic window. The biosensor was successfully applied for L-Dopa detection in human plasma, showing good recoveries, and preliminary investigations suggest its potential for quantifying L-Dopa in plant matrices as well.