Anti-inflammatory and early osteogenic potential of a 96% ethanol extract of Drynaria bonii.

ETHNOPHARMACOLOGICAL RELEVANCE: Drynaria bonii H. Christ rhizome is traditionally used in Vietnamese medicine for fractures and musculoskeletal disorders. However, the effects of extraction solvent on chemical composition and biological activities relevant to the inflammatory and early mineralization phases of bone repair remain insufficiently defined.

AIM OF THE STUDY: This study compared 30%, 70%, and 96% ethanol extracts of D. bonii (DB30, DB70, and DB96, respectively) to identify a lead extract for further fracture-healing-oriented investigation.

METHODS

Rhizome extracts prepared with 30%, 70%, and 96% ethanol were profiled by ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UHPLC-QTOF-MS/MS). TNF-α inhibition was evaluated in LPS-stimulated RAW264.7 macrophages; acute anti-inflammatory activity was assessed in carrageenan-induced mouse paw edema; and calcium-associated staining was evaluated in canine mesenchymal stem cell (MSC) cultures using Alizarin Red S (ARS). Acute oral toxicity was assessed at 5000 mg/kg, and selected constituents were isolated from DB96. The four NMR-characterized constituents were subsequently evaluated by molecular docking against IκB kinase β (IKKβ).

RESULTS

The extracts showed distinct qualitative LC-MS/MS profiles, with DB70 displaying a broader curated phenolic/flavonoid profile and DB96 showing a different compositional pattern. DB96 produced the strongest TNF-α inhibition (IC50 = 32.05 μg/mL), compared with DB70 (63.73 μg/mL), whereas DB30 did not reach 50% inhibition at 100 μg/mL. DB96 also showed the largest numerical paw-edema inhibition among the extract groups at 1 and 24 h; however, statistical comparisons were performed only against the disease-control group and not among active treatment groups. In canine MSC cultures, DB96 at 50 μg/mL showed the highest descriptive normalized ARS-positive area among the extracts (12.0 ± 2.1%), although staining remained weak relative to the positive control and did not establish complete osteogenic differentiation. Betulin, ursolic acid, rutin, and kaempferol were isolated from DB96 and independently identified by NMR from a preparative DB96 batch. Among these constituents, ursolic acid and betulin showed the most favorable predicted IKKβ docking profiles, with binding energies of -8.52 and -8.41 kcal/mol, respectively. No mortality or overt acute toxicity was observed for any of the three extracts after a single oral dose of 5000 mg/kg.

CONCLUSION

The findings support DB96 as the lead extract among the three preparations for subsequent mechanistic and bone-repair-oriented investigation. Docking provided complementary mechanistic support for possible interactions of selected DB96 constituents with IKKβ, but did not establish direct target inhibition. Direct fracture-healing efficacy, definitive osteogenic differentiation, quantitative constituent enrichment, and repeated-dose safety remain to be established.