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

Abstract

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). H2O2 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.