A double-duty champion: Eurycoma longifolia counters oxidative stress and unlocks autophagy to safeguard neurons and muscle cells

Objectives To investigate whether selected Malaysian plant extracts modulate the antioxidant-autophagy axis to enhance neuronal and skeletal muscle protection. Methods Four standardized extracts ( Persicaria minor, Eurycoma longifolia (EL) , Labisia pumila, and Ipomoea aquatica ) were evaluated for antioxidant activity at 100, 200, and 300 μg/mL using ABTS, DPPH, and ORAC assays. The most active extract was further examined in SH-SY5Y neuronal-like cells and differentiated C2C12 myotubes. Cell viability was assessed using CCK-8, oxidative DNA damage was measured by 8-OHdG ELISA, and autophagy-related changes were evaluated by LC3/p62 immunofluorescence and western blot analysis of LC3-II and p62 with chloroquine (CQ) as a lysosomal inhibitor. Results Among the four extracts, EL exhibited the strongest antioxidant activity, achieving the highest values in ABTS (107.59 ± 7.98 μmol Trolox/g), DPPH (27.28 ± 1.29 μmol Trolox/g), and ORAC (81.12 ± 4.81 μmol Trolox/g) at 300 μg/mL, also showing the lowest IC₅₀ (167.22, 97.64, and 67.90 μg/mL for ABTS, DPPH, and ORAC, respectively), with regression analyses confirming significant dose-response relationships (R² > 0.96, p < 0.05). CCK-8 analysis showed that EL at 100–300 μg/mL did not cause overt cytotoxicity in either cell model after 24 h treatment ( p > 0.2), whereas the positive cytotoxicity control markedly reduced viability ( p < 0.001). 8-OHdG ELISA showed that EL reduced oxidative DNA damage, with significant reductions at 200 and 300 μg/mL in both SH-SY5Y and C2C12 cells ( p < 0.05). H 2 O 2 markedly increased 8-OHdG levels in both models ( p < 0.001). Immunofluorescence analysis showed concentration-dependent modulation of LC3 and p62 autophagy-related markers. In SH-SY5Y cells, treatment with EL induced a dose-dependent increase in LC3 puncta ( p < 0.001) and a significant reduction in p62 fluorescence ( p < 0.01). Similar trends were observed in C2C12 myotubes, where LC3 puncta formation was enhanced ( p < 0.05), and p62 levels decreased ( p < 0.01). Western blot validation further showed that EL increased LC3-II abundance and that EL + CQ further increased LC3-II accumulation compared with CQ alone, particularly at 300 μg/mL ( p < 0.05). p62 decreased under basal EL treatment but showed a non-canonical decrease in the CQ-only group and was therefore interpreted cautiously. Conclusions EL showed the strongest antioxidant activity among the tested Malaysian plant extracts and modulated autophagy-related cellular markers without overt cytotoxicity. These findings identify EL as a promising candidate for further investigation in oxidative stress- and proteostasis-related cellular protection.