Thymoquinol-2-O-β-D-glucopyranoside: Isolation from Pulicaria Jaubertii, Anticancer Efficacy, and Comparative Apoptotic Markers Binding Studies with Thymoquinone and Thymoquinol

Thymoquinol-2-O-β-D-glucopyranoside (Th-Ol-G) isolated from Pulicaria jaubertii was characterized by 1D-(¹H, ¹³C, 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 (IC₅₀ = 13 ± 1.2 and 12 ± 0.6 µg/mL, respectively), compared to its effect on the noncarcinogenic HEK-293 cell (IC₅₀ 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.