Gracillin

Dioscoreae hypoglaucae rhizome (DHR) is the dried rhizome of Dioscorea hypoglauca polibin, which is rich in steroidal saponins. The steroidal saponins present in DHR exhibit great pharmacological activities, particularly in inflammatory and anti-hyperuricemia effects. Despite steroidal saponins are recognized as primary bioactive components in DHR, the enrichment and purification processes of steroidal saponins is rare. The research aimed to explore the purification process and anti-inflammatory activity of steroidal saponins of DHR. Firstly, the steroidal saponins in DHR were characterized by UPLC-Q/TOF-MS, and six steroidal saponins including protodioscin, protogracillin, pseudoprotodioscin, pseudoprotogtacillin, dioscin and gracillin were initially identified. Then, the steroidal saponins in DHR were purified by seven types of macroporous resins (D-101, S-8, AB-8, ADS-7, HPD-600, HPD-100, DM130), and HPD-100 resin with relative great static adsorption and desorption capacities was used for purification of steroidal saponins from DHR. The optimized purification parameters of HPD-100 resin were explored by static and dynamic adsorption and desorption experiments, and optimal purification conditions were loading flow rate 1.2 BV/h, loading concentration 0.1 g/mL, loading volume 3.5 BV, elution solvent 70 % (v/v) ethanol, elution speed 1.2 BV/h and elution volume 3 BV. The purity of steroidal saponins was increased by 5.78 times (from 6.93 ± 0.01 % to 40.07 ± 2.63 %) after HPD-100 resin purification. Furthermore, the anti-inflammatory activities of the crude extracts and purified steroidal saponins extracts of DHR were investigated by LPS-stimulated RAW264.7 macrophages inflammation model. The results demonstrated the purified steroidal saponins extracts of DHR exhibited better anti-inflammatory activity compared to the crude extracts of DHR. This study conducted a feasible and efficient HPD-100 resin method for purification of steroidal saponins from DHR, and also provided evidences for the development and utilization of steroidal saponins in DHR.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy with limited treatment options and poor patient survival. Despite the use of combination chemotherapy regimens, including FOLFIRINOX and gemcitabine-based therapies, the majority of patients experience relapse, underscoring the urgent need for novel therapeutic strategies. Gracillin, a natural triterpenoidal saponin, has demonstrated anticancer potential in various malignancies; however, its effects on PDAC and underlying molecular mechanisms remain unexplored. In this study, we provide the first comprehensive investigation of the anticancer properties of gracillin in PDAC. Our findings demonstrate that gracillin effectively inhibits PDAC cell viability, induces mitochondria-mediated apoptosis, and promotes oxidative damage by disrupting intracellular redox homeostasis. Mechanistically, gracillin exerts its effects by inhibiting the NRF2/HO-1 antioxidant axis, leading to excessive reactive oxygen species (ROS) accumulation and mitochondrial dysfunction. Molecular docking and molecular dynamics simulations further revealed a strong binding affinity between gracillin and NRF2, suggesting that gracillin may act as a direct NRF2 inhibitor. To further validate that anticancer effects of gracillin are mediated through NRF2 inhibition, we also assessed the impact of combining gracillin treatment with siRNA-mediated NRF2 silencing. Additionally, we found that gracillin enhances the cytotoxicity of gemcitabine, exhibiting a synergistic effect in PDAC cells. Finally, in vivo xenograft studies confirmed the tumor-suppressive effects of gracillin, further validating its potential as an effective therapeutic agent for PDAC. Overall, our study elucidates the potential of gracillin as a promising anticancer agent targeting the NRF2/HO-1 axis to disrupt redox homeostasis in PDAC. These findings provide new insights into the development of NRF2-targeted therapies for overcoming chemoresistance in PDAC.