Cassipourol and β-sitosterol from Malva parviflora L.: a mechanistic study of dual anti-inflammatory action against COX/LOX and TNF-α/BCL-2.

Although inflammation protects our bodies against harmful stimuli, uncontrolled inflammation drives serious chronic disorders. Malva parviflora L. (family Malvaceae) may represent a source for anti-inflammatory metabolites based on its potent anti-inflammatory activity. Its total ethanol extract demonstrated notable inhibition of cyclooxygenase-1/2 (COX-1/COX-2) and 5-lipoxygenase (5-LOX), with IC50 values comparable to those of the reference drugs indomethacin and zileuton, respectively. The hexane fraction was the most active fraction (lowest IC₅₀) among the solvent partitions (dichloromethane, ethyl acetate, and butanol). Subsequent column chromatography of the hexane fraction produced two compounds: Cassipourol, isolated for the first time from the Malvaceae family, and β-sitosterol. Their structures were confirmed by matching their NMR and mass spectrometry data with literature. Both were validated in vitro as dual COX/LOX inhibitors, exhibiting IC₅₀ values comparable to those of the standards. To uncover additional mechanisms, a compound-target-inflammation network was constructed using network pharmacology approaches, revealing 178 shared targets. Among these, tumor necrosis factor (TNF-α) and the antiapoptotic protein B-cell lymphoma 2 (BCL-2) emerged as central nodes linked to inflammatory pathways. Subsequent assays in human colon carcinoma (Caco-2) and lung adenocarcinoma (A549) cell lines showed that β-sitosterol suppressed TNF-α and BCL-2 by approximately 55%, whereas cassipourol displayed only modest inhibition (~20%). Molecular docking predicted moderate (ca. -4.5) and strong (> -5) binding affinities of both compounds to key inflammatory targets. Collectively, these results suggest that β-sitosterol from M. parviflora is a promising multitarget lead for inflammatory disorders, including cancer, whereas cassipourol requires further structural optimization and mechanistic investigation to improve its unfavorable physicochemical properties.