Levodopa in Mucuna pruriens and its degradation

Mucuna pruriens is the best known natural source of L-dopa, the gold standard for treatment of Parkinsonism. M. pruriens varieties are protein rich supplements, and are used as food and fodder worldwide. Here, we report L-dopa contents in seeds of fifty six accessions of four M. pruriens varieties, M. pruriens var. pruriens, M. pruriens var. hirsuta, M. pruriens var. utilis and M. pruriens var. thekkadiensis, quantified by HPTLC-densitometry. L-dopa contents varied between 0.58 to 6.42 (%, dr. wt.). High and low L-dopa yielding genotypes/chemotypes of M. pruriens could be multiplied for medicinal and nutritional purposes, respectively. HPTLC profiles of M. pruriens seeds on repeated extraction (24 h) in 1:1 formic acid-alcohol followed by development in butanol:acetic acid:water (4:1:1, v/v) showed consistent degradation of L-dopa (Rf 0.34 ± 0.02) into a second peak (Rf 0.41 ± 0.02). An average of 52.11% degradation of L-dopa was found in seeds of M. pruriens varieties. Since M. pruriens seeds and/or L-dopa are used for treatment of Parkinson’s disease and as an aphrodisiac both in modern and/or traditional systems of medicine, the finding of high level of L-dopa degradation (in pure form and in M. pruriens extracts) into damaging quinones and ROS is very significant.

products of L-dopa autoxidation are cytotoxic to cellular systems [22][23][24][25] . L-dopa is also an antinutritional factor and its consumption causes vomiting, nausea, abdominal distention, dyskinesia etc 26 . This is due to the conversion of L-dopa into dopamine in the peripheral nervous system by dopa decarboxylase. Moreover, M. pruriens var. pruriens and L-dopa could recover spermatogenic loss which makes them the treatment of choice for infertility 27,28 .
The degradation patterns of L-dopa in M. pruriens var. pruriens seed extracts and standard L-dopa in solvent media of 1:1 formic acid-alcohol (acidic), 20 mM Tris buffer (pH 7.2, neutral) and water at various time periods (1 h after initiation of extraction, 1, 7, 30 days after initiation of extraction) were tested through HPTLC profiling. Time dependent degradation was observed in M. pruriens var. pruriens extracts and in standard L-dopa at these pH values (Fig. S8-S19). The degradation rates of L-dopa in M. pruriens var. pruriens extracts and standard L-dopa were relatively low at 4 °C.
We consistently found degradation patterns of L-dopa in M. pruriens seeds in the acidic medium of 1:1 formic acid-alcohol. On HPTLC profiling, seed extracts of all four M. pruriens varieties (collected from various wild locations and EP grown) showed L-dopa at Rf 0.34 ± 0.02 and a consistent second degradation peak at Rf 0.41 ± 0.02. This degraded peak was indentified as a labile mix of dopamine, dopachrome, leucodopachrome, dopaquinone and other ROS by HPTLC, DART-MS and LC/EI-MS. On an average, the degradation of L-dopa in a 24 h extraction protocol was a high 52.11%, which is significant enough to cause adverse effects in biological systems.
HPTLC profiles of M. pruriens var. pruriens seed extract (4450) and L-dopa standard in acidic, neutral and water media showed time dependent degradation. L-dopa degradation rates in the acidic medium (1:1 formic acid-alcohol) were equivalent or even higher compared to Tris buffer (neutral) or water under identical extraction periods. In seven days, M. pruriens var. pruriens seed extract and L-dopa in these liquid media resulted in degradation with gradual appearance of a black deposit. In 30 days, both M. pruriens var. pruriens seed extract and L-dopa in these solvent media resulted in significant degradation and strong black deposits. The degradation rates of L-dopa in M. pruriens var. pruriens extracts and standard L-dopa were low at 4 °C compared to room temperature.
Recent studies showed that L-dopa degradation products and dopamine adducts result in oxidative stress and cause selective cytotoxicity of neuronal cells inducing pathogenesis in PD 18,[20][21][22][23][24][25]43,44 . Moreover, in PD treatment, M. pruriens seeds or L-dopa are administered for very long periods. Chronic L-dopa therapy in PD results in movement disorders or dyskinesia in most patients. M. pruriens seeds and its preparations are also used for the treatment of PD in the traditional medicinal system of Ayurveda since ancient times. Certain studies claimed that M. pruriens seeds are even more effective than L-dopa in PD treatment. Some literature reports caution insufficient evidence to recommend the clinical use of M. pruriens in the treatment of PD 45,46 . M. pruriens seeds are also used in tonics for male vitality and virility in Ayurveda 28,47 .
Since M. pruriens seeds and/or L-dopa are used for treatment of PD and as an aphrodisiac both in modern and/or traditional systems of medicine, the finding of high level of L-dopa degradation (in its pure form and in M. pruriens extracts) into damaging quinones and ROS is very significant. Our finding of consistent degradation products suggests the need for careful review of the processing of M. pruriens seeds (drying/roasting, powdering, extraction), L-dopa and their mode(s) of administration (medium, pH, temperature). Further studies are required to confirm the adverse effects of the degradation products from the 'cure' (M. pruriens, L-dopa) itself in PD patients and other users.  (Table 1). GPS coordinates and other pertinent field data of these M. pruriens accessions were recorded during field trips. Voucher specimens of these M. pruriens accessions were deposited at the Herbarium (TBGT) of Jawaharlal Nehru Tropical Botanic Garden and Research Institute (JNTBGRI). Seeds of these thirty M. pruriens accessions collected were dried and powdered (separately).
A L-dopa extraction. M. pruriens seed powder (2 g each) was extracted with 1:1 formic acid-alcohol (20 ml, 2 h) at room temperature and the extract was filtered. Seed powder residue was then repeatedly extracted with 1:1 formic acid-alcohol (3 × 10 ml, 2 h each), and extracts were filtered. This seed residue was again extracted with 1:1 formic acid-alcohol (10 ml, overnight). Filtrates (of five extractions) were pooled, centrifuged (5000 rpm, 30 min, 10 °C) and made up to 100 ml using 1:1 formic acid-alcohol. This M. pruriens seed extract (5 ml) was concentrated on a rotary evaporator, and the extract weight was recorded. This (concentrated) M. pruriens seed extract was dissolved in 20 ml 1:1 formic acid-alcohol and used for L-dopa quantification by HPTLC-densitometry. This extraction protocol was followed for quantification of L-dopa in seeds of all (56) M. pruriens accessions (Table 1). Extraction protocol was optimized for 24 h and cold extraction was preferred (against hot extraction) due to the labile nature (degradation) of L-dopa during extraction.
Data analysis, validation. HPTLC-based quantification of L-dopa was validated in terms of precision, accuracy, repeatability and linearity. Specificity of the assays was tested by repeated application of standard L-dopa. Rf values (Rf 0.34 ± 0.02, n = 56) of the standard was reproducible, and was found to be same as the values observed for the peak (L-dopa) in M. pruriens seed extracts. Calibration curve was plotted between amount of standard L-dopa (fresh) versus average response (peak area) (y = 6.542x + 88.22, R 2 = 0.996). Linearity of the calibration curve in the range 100-1000 ng was ensured. Percentage L-dopa content(s) (Rf 0.34 ± 0.02, % ± SD, n = 6, based on dry weight) in M. pruriens extracts were calculated from peak areas using the standard curve. Percentage of second degradation peak (SDP, Rf 0.41 ± 0.02) which is a combination of labile molecules was also quantified based on L-dopa standard curve. Repeatability of sample application (instrumental precision) was assessed by applying a sample solution (M. pruriens extract, 4 μ l) on a HPTLC plate developed up to 80 mm under saturation conditions with butanol:acetic acid:water (4:1:1, v/v) as the mobile phase in the twin-trough glass chamber (previously saturated with the solvent for 30 min). The spot (L-dopa) was scanned six times, % coefficient of variation was acceptable. Robustness of the method was checked by slightly altering the mobile phase composition and plate developing distance was checked. No considerable effect on the data was found.