Thymoquinol‐2‐ <i>O</i> ‐ <i>β</i> ‐D‐glucopyranoside: Isolation from <i>Pulicaria Jaubertii</i> , Anticancer Efficacy, and Comparative Apoptotic Markers Binding Studies with Thymoquinone and Thymoquinol

Abstract Thymoquinol‐2‐ O ‐ β ‐D‐glucopyranoside (Th‐Ol‐G) isolated from Pulicaria jaubertii was characterized by 1D‐( 1 H, 13 C, DEPT) and 2D‐(COSY, HSQC, HMBC) NMR analyses. This study provides new findings for the anticancer activity of Th‐Ol‐G and compares its silico binding affinity to caspases 3 and 9 with its corresponding aglycones, thymoquinone (Th‐One) and thymoquinol (Th‐Ol). Th‐Ol‐G has exerted marked cytotoxicity against HCT‐116 and HeLa cells (IC 50 = 13 ± 1.2 and 12 ± 0.6 µg/mL, respectively), compared to its effect on the noncarcinogenic HEK‐293 cell (IC 50 value of 22 ± 1.3 µg/mL). Th‐Ol‐G induced apoptosis in cancer cells evident by significant shrinkage of nuclei in DAPI‐stained cells compared to control. Th‐Ol‐G markedly increased the expression of apoptotic markers, caspases 3 and 9 in HeLa and HCT‐116 cells compared to untreated cells. Th‐Ol‐G has demonstrated a marked lower docking score of −8.8 and −6.3 kcal/mol compared to Th‐One (−3.8 and −3.2 kcal/mol) and Th‐Ol (−4.0 and −4.2 kcal/mol) with MM‐GBSA binding free energy values of −49.1 and −26.3 kcal/mol for caspase 3 and 9, respectively. Theoretical findings suggest that Th‐Ol‐G exhibits stability and strong binding to both enzymes, with the sugar moiety engaging in H‐bonding with the enzyme's amino acids. Additionally, conformational changes in caspase 9′s binding cavity indicate Th‐Ol‐G's stable interaction with the protein.