Detection And Quantification Of Levodopa (L-Dopa) Content In Lycoperdon SP., Xylatia SP. And Physarum Polycephalum

Abstract

L-DOPA is an amino acid that is clinically used to treat Parkinson's disease. Even though synthetic L-DOPA is effective and in high demand, some severe side effects have been reported from patients on long-term L-DOPA medication. However, direct consumption of L-DOPA of rich biomass instead chemically synthesized L-DOPA has been found to eliminate the side-effect. The objectives of this study were to detect the presence and measure L-DOPA contents in different microbial groups including slime molds (Physarum polycephalum), puffballs (Lycoperdon perlatum), Xylaria (Xylaria polipmorpha and Xylaria sp.) by thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC). The research also initially investigated if the L-DOPA content of a certain fungus would be different at different stages (fruiting body and mycelia) in its life cycle. To initially detect the presence of L-DOPA, the TLC method was applied with the homogenate obtained from formic acid 2% extraction. Silica gel plate 60 F254 (Merck PGaA, Germany) activated in methanol at 60oC for 5 minutes was used as the stationary phase, and the solvent mixture of isopropanol: ethyl acetate: water: glacial acetic acid (29:19:10:1) was used as the mobile phase. The visualization was done with ninhydrin dye and heated at 105oC for 5 minutes. Standard L-DOPA was used as the reference. The obtained results suggested the presence of L-DOPA in all the tested samples. HPLC-UV quantification found that the L-DOPA contents were different among the studied species. Specifically, P. polycephalum microplasmodia had the highest content (1.09 mg/g), followed by the fruiting bodies of L. perlatum (0.34 mg/g) and X. polimorpha (0.22 mg/g). Comparing between different stages of puffball and Xylaria: the young fruiting bodies of L. perlatum, X. polimorpha and Xylaria sp contained 0.34 (mg/g), 0.22 (mg/g) and 0.23 (mg/g) respectively; and those of their mycelial was 0.29 (mg/g), 0.06 (mg/g) and 0.34 (mg/g). These revealed that different stages in the life cycle would influence the L-DOPA content, but the trend is largely dependent on each species. Specifically, only the trophic state (mycelia) of Xylaria sp. produced a higher amount of L-DOPA that that of the reproductive stage (fruiting body), but the rest is either comparable or lower.