Integrated AUF-UPLC-MS and fraction-activity correlation to identify COX-2-targeted constituents of Shaoyao Gancao decoction

Abstract

Shaoyao Gancao Decoction (SGD), a classical multi-ingredient herbal formula, exerts potent anti-inflammatory and anti-rheumatoid arthritis effects against rheumatoid arthritis (RA). Existing cellular experiments have proven that SGD can downregulate Cyclooxygenase-2 (COX-2) expression in inflammatory macrophages, implying a potential link between its anti-arthritic efficacy and COX-2 modulation. Nevertheless, its constituents with potential COX-2 binding capacity and corresponding anti-RA material basis are unclear, hindering its pharmaceutical exploitation. In this study, an integrated strategy combining affinity ultrafiltration-UPLC-MS screening, fraction-activity correlation modeling, molecular simulation and in vitro enzyme assays was constructed to characterize COX-2-interacting components from SGD. Nineteen COX-2-binding constituents were captured and characterized, mainly paeoniflorin, glycyrrhizic acid and licorice glycoside B. Derived from Bai Shao (BS) and Zhi Gancao (ZGC), these compounds cover monoterpenoid glycosides, triterpenoid saponins, flavonoids and fatty acids, embodying SGD’s multi-component feature. Molecular docking results predicted favorable binding interactions, and molecular dynamics simulations further demonstrated that core monomers could form stable COX-2 binding complexes. These core components stably bound COX-2 via persistent hydrogen bonds. In vitro enzyme tests confirmed paeoniflorin, albiflorin, glycyrrhizic acid and glycyrrhetinic acid strongly suppressed COX-2 with IC 50 of 23.8, 53.2, 54.1 and 58.9 μM, respectively. Among serial gradient eluates (10%–70% gradient) of BS, the 30% ethanol fraction exhibited the most potent COX-2 inhibitory activity (IC 50 = 27.63 μg/mL). Further enrichment via 40% ethanol macroporous separation yielded a refined BS fraction with superior activity (IC 50 = 23.8 μg/mL). Cell assays on LPS-induced RAW264.7 macrophages indicated the reconstituted active fraction of Shaoyao Gancao Decoction (SGF) inhibited NO release more effectively than crude SGD extract. This study established a robust workflow coupling target screening, chemical identification, computational validation and active fraction enrichment for complex herbal extracts. This work screened potential COX-2-binding constituents from SGD using analytical chemistry workflows and verified the COX-2-inhibitory potency of four monomers via in-vitro enzyme assays. Prior cellular evidence supports that SGD suppresses COX-2 in LPS-stimulated macrophages, suggesting a link between its anti-inflammatory action and COX-2 modulation. However, systematic in vivo RA animal studies are needed to validate the COX-2-dependent anti-RA mechanism of SGD, which lies outside the analytical scope of the present investigation.