Physiological microspecies-constrained docking links UHPLC–MS/MS fingerprints to multi-target COX-1/COX-2/5-LOX inhibition by an ultrasound extract of Ziziphus lotus fruits

Abstract

Ziziphus lotus (L.) Desf. fruits are traditionally used against inflammatory disorders, yet the bioactive phytochemical forms present at physiological pH are rarely considered in molecular docking, leading to misleading target–ligand hypotheses. Here, an ultrasound-assisted hydroethanolic extraction was optimized to obtain a phenolic-rich extract (yield 47.03 ± 2.26%; TPC 19.72 mg GAE g−1; TFC 9.30 mg QE g−1). UHPLC-MS/MS revealed rutin (42.05% AUC), delphinidin-3-rutinoside (21.30% AUC) and kaempferol glycosides (8.47% AUC) as major constituents. The extract inhibited 5-lipoxygenase, COX-1 and COX-2 with IC50 values of 158.35 ± 5.33, 89.78 ± 1.63 and 92.96 ± 2.22 µg mL−1, respectively. To address a key gap in in silico anti-inflammatory studies, we implemented pH-aware microspecies selection: dominant protonation states at pH 7.4 were predicted and docked into 5-LOX and COX isoforms. Rutin and delphinidin-3-rutinoside showed the most favorable binding energies (≈−8.2 and −8.1 kcal mol−1) with stable hydrogen-bond and π-interactions, supporting multi-target inhibition. This integrated pH-constrained workflow strengthens the translation of docking to biologically relevant chemical space.