*2.3. Plant Extraction*

The seeds of *M. pruriens* (10 kg) were roasted by a roasting drum for 30 min at a temperature of 180 ◦C. The roasting temperature was measured by a sensor located in the drum. The roasted seeds were crushed to fine powders by passing through a stainless-steel sieve with a nominal mesh aperture of 180 μm. The fine powders were extracted with boiling water at 100 ◦C. The ratio of fine powder to hot water was 1:7. The water extract was mixed using the heating and stirring method with the heater and homogenizer for 15 min. After filtration with the white cloth, the filtrate was then freeze-dried to remove the solvent for 24 h. Finally, the concentrate crude extract was obtained.

#### *2.4. HPLC Analysis of L-Dopa in M. pruriens Extract*

Isocratic HPLC analysis was performed to analyze the amount of L-dopa remaining in the extract using Perkin Elmer series 200, USA, equipped with auto-sampler and a UV–Vis detector. The injection volume was 20 μL for all samples. The mobile phase for elution was 0.1 M KH2PO4 (pH 2.5). Samples were eluted on ACE-129-2546 (250 mm × 4.6 mm) C18 column with a flow rate of 1.0 mL/min. L-dopa peak was integrated at the wavelength of 283 nm. A stock solution of L-dopa was prepared to obtain L-dopa concentration of 1 mg/mL. The stock solution was diluted to 10, 20, 30, 40, and 50 μg/mL in 0.1 N HCl. *M. pruriens* aqueous extract was weighed and dissolved in 0.1 N HCl. Samples were then filtered through Whatman filter paper and 0.45 μm membrane filter.

#### *2.5. Quantitative Analysis of Total Phenolic Content*

Total phenolic content in *M. pruriens* seed extract was determined by the Folin– Ciocalteu reaction. Gallic acid solution (3.9–125 μg/mL) and *M. pruriens* seed extract solution (2 mg/mL) were mixed with 10% *v/v* of Folin–Ciocalteu phenol reagent (100 μL) for 1 min. After 4 min of incubation, 7.5% *w/v* Na2CO3 solution (50 μL) was added, and the mixture was further incubated in the dark at room temperature for 2 h. The absorbance was read using a UV–visible spectrophotometer (Spectramax M3, Thermo Scientific, Waltham, MA, USA) at a wavelength of 765 nm. Total phenolic contents were calculated from the gallic acid standard curve. Data were expressed as mg/g gallic acid equivalents (GAE) of dry crude extract.

#### *2.6. Quantitative Analysis of Total Flavonoid Content*

The total flavonoid content in *M. pruriens* seed extract was determined by the aluminum chloride colorimetric method. Quercetin solution (3.9–500 μg/mL) and *M. pruriens* seed extract solution (2 mg/mL, 100 μL/well) were mixed with 5% NaNO2 (30 μL) and incubated for 5 min. Aluminum chloride (2% *w/v*, 50 μL) was added and incubated for

6 min followed by 10 min of incubation with 1 N NaOH (50 μL). The absorbance was measured at a wavelength of 510 nm with a UV–Vis spectrophotometer (Spectramax M3, Thermo Scientific, Waltham, MA, USA). Total flavonoid contents were calculated from quercetin and were expressed as mg/g quercetin equivalents (QE) of dry crude extract.
