*3.2. Total Phenolic and Total Flavonoid Content in M. pruriens Aqueous Extract*

The gallic acid standard curve equation was expressed as y = 0.0173x + 0.1011, r2 = 0.999. The average total phenolic content in the extract was 105.59 ± 0.84 mg gallic acid equivalent (GAE)/g crude dry extract. The total flavonoid content in *M. pruriens* seed extract determined by the aluminum chloride colorimetric method was expressed in quercetin equivalent amounts. The quercetin standard curve equation was y = 0.0013x + 0.0481, r2 = 0.9977. The average total flavonoid content in the extract was 80.74 ± 0.51 mg quercetin equivalent (QE)/g crude dry extract.

#### *3.3. Cell Viability Assay*

P19-derived neurons culture was treated with an increasing concentration of *M. pruriens* seed aqueous extract and L-Dopa (0–10 μg/mL). The neuronal viability was determined by XTT assay. The results were expressed as % cell viability. The effective concentration of *M. pruriens* seed aqueous extract at 10 ng/mL (% neuron viability = 114.48 ± 19.25%) that enhanced the survival of cultured neurons more than control (% neuron viability of the control = 100.47 ± 0.67%) were further investigated for the neuroprotective ability (Figure 1). Interestingly, L-dopa showed no enhancing cell viability effect on P19-derived neurons, suggesting that L-dopa was not responsible for the neuroprotective ability of *M. pruriens*. Therefore, only the extract was further investigated for its neuroprotective ability.

**Figure 1.** P19-derived neurons cell viability in various concentration (1–10,000 ng/mL) of *Mucuna pruriens* seed aqueous extract and L-dopa.

#### *3.4. Serum Deprivation Method*

P19-derived neurons culture was treated with 10 ng/mL of *M. pruriens* seed aqueous extract compared with toxic condition (0.5% *v/v* DMSO in α-MEM without serum) and positive control (1 nM quercetin). Cell viability was assayed by XTT reduction method. The results show significant cell viability in 10 ng/mL Mucuna (28.73 ± 1.90%) compared with the toxic condition (8.37 ± 0.73%) (Figure 2). The % neuron viability, when treated with 1 nM quercetin (positive control), was 34.73 ± 3.93%. No significant difference was found between the % neuron viability when treated with 10 ng/mL Mucuna and 1 nM quercetin.
