Comparative evaluation of in vitro biological activities of chemically characterized tilia species: Protective effects against inflammation and cytotoxic effects on pancreatic cancer cells with 2d and 3d spheroid models

Cancer is the second leading cause of death worldwide. The most common methods for cancer treatment are radiation therapy and chemotherapy; however, these methods adversely affect the healthy cells. Medicinal plants could be safer alternatives with rich secondary metabolites having anti-inflammatory and anticancer properties for decreasing the risk of oxidative stress-related diseases including inflammatory conditions and cancer. This study aims to comparatively assess the hydroalcoholic extracts of chemically characterized Tilia species in terms of their antioxidant activities, protective effects against inflammation, and anticancer potentials. For the antioxidant activities DPPH, CUPRAC, and FRAP assays were performed. The protective role against inflammation was evaluated by in vitro nitrite assay in LPS-induced RAW 264.7 murine macrophage cells and IL-6 releasing and PGE2 production inhibition assays. Anticancer activities of the extracts were assessed with a 2D cytotoxicity assay and 3D spheroid formation/growth assay towards MIA PaCa-2 pancreatic cancer cell line. The highest phenolic and flavonoid contents were determined in T. rubra flowers and bracts which showed the highest antioxidant activities in FRAP, CUPRAC and DPPH assays. All extracts inhibited LPS-induced nitrite production by more than 50 % at 0.125–1 mg/mL concentrations range. T. tomentosa bracts exerted the best anti-inflammatory activity with 90% NO inhibition. In addition, all Tilia species exerted potent reducing capacity on IL-6 releasing and PGE2 production. Bracts, flowers, and inflorescences extracts of T. rubra exerted the highest cytotoxic activity with IC50 values of 0.16 ± 0.01, 0.21 ± 0.01, and 0.24 ± 0.05 mg/mL, respectively in 2D cytotoxicity assay. Similarly, bracts of T. rubra showed the strongest spheroid size change with 50 % reduction with the 3D spheroid assay. In conclusion, our results suggest that all investigated Tilia species showed potent protective activity against inflammation and especially T. rubra flowers and bracts have shown promising anticancer activity against MIA PaCa-3 pancreatic cancer cell line. Keywords: Antioxidant activity, anti-inflammatory activity, anticancer activity, Tilia species, 3D spheroid model