Chemical characterization of the ORY mixture. A list of the identified components is reported in the following: cycloartenyl ferulate (CAF, 37%), 24–methylenecycloartanyl ferulate (24–MET, 40%), campesteryl ferulate (CAM, 11%), and β–sitosteryl ferulates (SIT, 12%).

Gamma-oryzanol promotes neuronal differentiation in SH-SY5Y. SH-SY5Y is treated with a vehicle (CTRL), 10 µM retinoic acid (RA), or 1, 5, or 10 μg/mL gamma-oryzanol (ORY) for 5 days. (A) Representative confocal microscope images of SH-SY5Y labelled with β III-tubulin (red) and co-labelled with DAPI (blue). Magnification 63X, Scale bar = 10 µm. (B) The percentage of cells with neurites (C) and the number of cells per field were calculated in all the conditions respect to the control group. **** p < 0.0001; *** p < 0.001; ** p < 0.01; and * p < 0.05.

Gamma-oryzanol promotes neurotrophic effects in SH-SY5Y. Gene expression analysis of GAP43 (A), TrkB (B), and BDNF (C) normalized to the internal standard control gene (GAPDH). Data are represented as means ± S.E.M. of n = 3 experiments run in triplicate. One-way ANOVA tests with the Bonferroni post-test were used. **** p < 0.0001; *** p < 0.001; ** p < 0.01; and * p < 0.05.

Gamma-oryzanol positively affects neuronal differentiation of ahNPCs. AhNPCs are seeded in differentiating conditions in the presence of a vehicle (DMSO 0.2%) or gamma-oryzanol (ORY) at 1, 5, or 10 μg/mL for 24 h. (A) Percentage of MAP2+ cells over total viable cells. (B) Percentage of GFAP+ cells over total viable cells. (C) Quantification of apoptotic cells calculated over the total number of Hoechst+ nuclei. (D–G) Representative confocal microscope images of MAP2+ (red, (D,E)) and GFAP+ (green, (F,G)) cells derived from ahNPCs differentiated in the presence of a vehicle (veh, (D,F)) or ORY (10 μg/mL, (E,G)). Nuclei are stained with Hoechst (blue). Magnification 63X, Scale bar = 20 µm. Data are represented as means ± SD of n = 3 experiments run in triplicate. Student’s t-test. *** p < 0.001; ** p < 0.01; * p < 0.05 vs. DMSO-treated cells.

Gamma-oryzanol positively regulates nervous system development in zebrafish. (A–F) Representative images of the head region of 2 dpf Tg (-3.1neurog1:GFP) zebrafish embryos injected with std–MO or nrf2–MOs and then with DMSO or ORY: (A) std–MO + DMSO (n = 11); (B) nrf2a–MO + DMSO (n = 10); (C) nrf2b–MO + DMSO (n = 10); (D) std–MO + ORY (n = 18); (E) nrf2a–MO + ORY(n = 11); (F) nrf2b–MO + ORY (n = 10). (G) Quantification of the GFP fluorescence intensity of std–MO or nrf2–MOs embryos injected with DMSO or ORY. (H–M) islet1 whole mount in situ hybridization of std–MO or nrf2–MOs embryos injected with DMSO or ORY: (H) std–MO + DMSO (n = 17); (I) nrf2a–MO + DMSO(n = 15); (J) nrf2b–MO + DMSO (n = 14); (K) std–MO + ORY (n = 15); (L) nrf2a–MO + ORY (n = 18); (M) nrf2b–MO + ORY (n = 11). (N) Subdivision of std–MO or nrf2–MOs embryos injected with DMSO or ORY according to the relative islet1 expression. (O,P) RT-qPCR expression analyses of (O) gstp1 and (P) bdnf. Scale bar indicates 100 μm. Data are presented as means ± standard error of the mean (S.E.M.). One–Way ANOVA with Tukey post–hoc. *** p < 0.001; ** p < 0.01; * p < 0.05; ns: not significant.

Interaction motif predicted by molecular docking for 24–methylenecycloartanyl ferulate (green), one of the components of ORY, with Keap1 (A): the compounds inserted below the hot spots of the Keap1 cavity (24–methylenecycloartanyl ferulate is depicted as a representative example). Calculated binding energy values retrieved for the studied compounds and ferulic acid were introduced as references (B). Detailed view of the binding site of Keap1: 24–methylenecycloartanyl ferulate is depicted in green (C). Interacting residues (<5 Å) have been labeled.