In vitro and plant-based LC-MS/MS assessment of guggulsterone E and Z in Commiphora wightii (Arnott.) bhandari: A sustainable source of phytosteroids and antioxidants

• Altered sonication time and solvent ratio increase guggulsterone yield. • The study enables rapid and accurate detection of G-E and G-Z • MS/MS fragmentation studies validate G-E and G-Z in the samples. • In vitro callus has high flavonoid, phenolic content than plants. • Correlation studies shows G-E, G-Z links to metabolites and antioxidant activity. Commiphora wightii , a medicinal plant native to India, is renowned for its therapeutic properties, primarily due to the presence of the phytosteroids guggulsterone E (G-E) and guggulsterone Z (G-Z). However, excessive extraction of these compounds from the oleo-gum resin has placed the plant in the endangered category. This study explores plant cell culture techniques to generate in vitro callus cultures as an alternative source of these valuable phytosteroids. The highest biomass yield (4.84 g/50 ml FW and 0.193 g/50 ml DW) was obtained using Gamborg's B5 medium, supplemented with 2 mg/L 2,4-D and 0.5 mg/L kinetin. UPLC-ESI-Q-TOF-MS analysis confirmed the presence of G-E and G-Z in both the callus and oleo-gum resin, with characteristic parent and product ion peaks. Quantitative analysis revealed G-E and G-Z levels in callus at 28.90 ± 2.4 μg/g and 92.85 ± 7.7 μg/g, significantly lower than in the resin. However, callus exhibited higher antioxidant properties, including 2.35-fold increased flavonoid content, 1.60-fold higher phenolic content, and a 4.86-fold rise in ascorbic acid. These findings suggest that C. wightii callus cultures offer a promising alternative for guggulsterone production and antioxidant therapy.