Plant materials

Seeds of thirty accessions of M. pruriens varieties viz. M. pruriens var. pruriens (21), M. pruriens var. hirsuta (3), M. pruriens var. utilis (5) and M. pruriens var. thekkadiensis (1) were collected in January to April 2009 from various wild locations in Kerala in south India (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 second set of M. pruriens seeds collected were initially planted in a FGB of the species established at JNTBGRI. First generation seeds of 21 accessions of M. pruriens var. pruriens and 5 accessions of M. pruriens var. utilis were collected from the FGB and planted in the EP in Randomized Block Design with two replications of each accession and one plant in each replication. M. pruriens var. hirsuta and M. pruriens var. thekkadiensis accessions did not produce fruits in the FGB. Therefore, these two varieties were not planted in the EP. M. pruriens var. pruriens and M. pruriens var. utilis accessions were maintained in the EP in uniform conditions. M. pruriens accessions planted in both FGB and EP were irrigated as and when required and not supplemented with any external fertilizers. Second generation seeds of 21 accessions of M. pruriens var. pruriens and 5 accessions of M. pruriens var. utilis were collected in January to April 2011, dried and powdered (separately) (Table 1).

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.

Quantification of L-dopa

L-dopa content in M. pruriens seed extracts was quantified using an HPTLC (CAMAG, Switzerland) made up of Linomat V sample applicator, twin-trough plate development chamber, TLC Scanner 3 and WinCATS Software 4.03. M. pruriens seed extract (5 ml concentrated seed extract) was dissolved in 20 ml of 1:1 formic acid-alcohol (see L-dopa extraction), 4 μl of this solution was repeatedly applied to silica gel HPTLC plate (60 F254, E. Merck, Germany, 20 × 10 cm, 0.2 mm thickness) as 6 mm wide bands with Camag Linomat V sample applicator, fitted with a microsyringe, in N 2 flow (application rate −50 nL/s, space between two bands −11.3 mm, slit dimension- 6 × 0.45 mm, scanning speed −20 mm/s). L-dopa standard was also applied along with M. pruriens seed extracts. HPTLC plate was developed upto 80 mm in the twin-trough glass chamber pre-saturated for 30 min with mobile phase butanol:acetic acid:water (4:1:1, v/v, 24 ml). Developed plate was scanned densitometrically at 282 nm (deuterium lamp) using TLC Scanner 3 equipped with WinCATS software. L-dopa at Rf 0.34 ± 0.02 (n = 56) and a second degradation peak (SDP) at Rf 0.41 ± 0.02 (n = 56) were found in M. pruriens seed extract in butanol:acetic acid:water (4:1:1, v/v) (Fig. 2a–d). Similar quantification protocol was followed for all M. pruriens seed extracts. Freshly dissolved L-dopa did not show the second signal, but after 24 h in solvent (1:1 formic acid-alcohol) it showed a clear second signal at Rf 0.41 (Fig. 2a,b). Other signals in M. pruriens seed extracts were well resolved from these two L-dopa based signals (Fig. 2c,d). Solvent systems such as 7:3 ethanol:water, 4:1:1 butanol:acetic acid:water, 4:1:5 butanol:acetic acid:water and 4:2:1 butanol:acetic acid:water were tried for development of plates. Of these, 4:1:1 butanol:acetic acid:water gave best resolution of signals on development.

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, R2 = 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 μl) 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. Recovery studies were carried out (in two modes) by the addition of L-dopa to pre-analyzed M. pruriens extracts and they were again analyzed (see Quantification of L-dopa). In the first mode, (i) M. pruriens var. pruriens seed powder (Acc. No. 4088, 2 g) was extracted in the five-step protocol (24 h) and (ii) standard L-dopa (10 mg) was added initially to M. pruriens var. pruriens seed powder (Acc. No. 4088, 2 g) and extracted in the five-step protocol (24 h). (i), (ii) and (iii) fresh standard L-dopa (1 μg/μl), in 1:1 formic acid-alcohol were loaded (4 μl each) onto HPTLC plate, developed with butanol:acetic acid:water (4:1:1, v/v) and peak areas were measured at 282 nm (see L-dopa extraction, Quantification of L-dopa). % recovery of L-dopa was calculated from peak areas as 49.78%. In the second mode, M. pruriens var. pruriens seed powder (Acc. No. 4502, 2 g) was extracted in the standard five-step protocol (24 h). (i) M. pruriens var. pruriens extract in 1:1 formic acid-alcohol (4 μl), (ii) M. pruriens var. pruriens extract in 1:1 formic acid-alcohol (4 μl) and fresh L-dopa dissolved in 1:1 formic acid-alcohol (1 μg/μl, 4 μl) and (iii) fresh L-dopa dissolved in 1:1 formic acid-alcohol (1 μg/μl, 4 μl) were loaded onto HPTLC plate ((ii) co-spotted), developed and peak areas were measured. % L-dopa recovery was calculated as 99.30%. % residual standard deviations (RSD) were determined as 2.63 (L-dopa) and 3.58 (SDP). Limit of detection (LOD, average of 3.3 × SD of peak area/slope of calibration curve for 56 accessions, n = 6) and limit of quantification (LOQ, average of 10 × SD of peak area/slope of calibration curve for 56 accessions, n = 6) were determined for both L-dopa (LOD −31.46 ng, LOQ −95.32 ng) and SDP (LOD −21.10 ng, LOQ −63.93 ng)48,49.

L-dopa degradation

M. pruriens var. pruriens seed extract (Acc. No. 4450) prepared by the five stage extraction for 24 h (fresh M. pruriens var. pruriens seed extract) and standard L-dopa (fresh) were suspended in 1:1 formic acid-alcohol and kept at room temperature for seven days with occasional stirring. These resulted in decomposed M. pruriens var. pruriens seed extract and decomposed L-dopa standard, respectively. Fresh M. pruriens var. pruriens seed extract, freshly prepared L-dopa standard (both in 1:1 formic acid-alcohol), decomposed M. pruriens var. pruriens seed extract and decomposed L-dopa standard were applied onto silica gel plates (60 F254, E. Merck, Germany, 20 × 10 cm, 0.2 mm thickness) by HPTLC (CAMAG, Switzerland), developed in butanol:acetic acid:water (4:1:1, v/v, 24 ml) and scanned at 282 nm (TLC Scanner 3, CAMAG, Switzerland) (Fig. 2).

Fresh M. pruriens var. pruriens seed extract (100 mg), decomposed M. pruriens var. pruriens seed extract (100 mg) and decomposed L-dopa standard (26.1 mg) were analyzed by DART-MS on an AccuTOF JMS-T100LC Mass Spectrometer having a DART (JEOL, USA). Samples were analyzed directly in front of the DART source. Dry He was used at a flow rate of 4 LPM for ionization at 350 °C. Orifice 1 was set at 28 V, spectra were collected and the data from 6-8 scans were averaged (Fig. S1-S3). Again, decomposed M. pruriens var. pruriens seed extract (80 mg) and decomposed L-dopa standard (20 mg) were subjected to LC/ESI-MS analysis on a Surveyor-LCQ Deca XP plus system (Thermo Finnigan, USA) with Hypersil BDS C18 column (length 250 mm, int. dia. 4.6 mm, particle size 5 μm), mobile phase: methanol-water 85:15, inj. vol.: 10 μl, flow rate: 0.3 ml/min, run time: 30 min, LC detection: PDA/UV detector, 280 nm and mass detection: electro spray ionization (Fig. S4-S7).

Effect of pH on L-dopa degradation

M. pruriens var. pruriens (accession number 4450) seeds (1 g each) were separately extracted with 20 ml (each) of 1:1 formic acid-alcohol (strongly acidic), 20 mM Tris-HCl, 20 mM KCl (pH 7.2, neutral) and water at room temperature and at 4 °C. Similarly, standard L-dopa (5 mg) was extracted (dissolved) in 10 ml each of these three solvents at room temperature and at 4 °C. These M. pruriens var. pruriens seed/L-dopa extracts (3 μl each) were profiled using HPTLC-densitometry (as described in Quantification of L-dopa) at various time periods viz., 1 h after initiation of extraction, 1, 7 and 30 days after initiation of extraction (Fig. S8-S19).