Optimization of the Isolation and Purification of Formononetin from Lycopus lucidus Turcz. var. hirtus Regel, Structural Identification, and Its In Vitro Anti-Inhibitory Effects in TNF-α-Induced HFLS-RA Cells

Lycopus lucidus Turcz. var. hirtus Regel (L. lucidus var. hirtus) is a medicinal herb traditionally used in Chinese folk medicine for the treatment of inflammatory disorders, joint pain, and rheumatoid arthritis (RA)-like symptoms. However, systematic research on the bioactive flavonoids of this herb in RA-related cellular models is limited. The aim of this study was to isolate the main flavonoid from this herb and evaluate its in vitro anti-inhibitory effects in TNF-α-induced human RA fibroblast-like synoviocytes (HFLS-RA). Flavonoids were extracted, purified, and isolated using chromatographic techniques. The structure was elucidated by spectroscopic analysis. The effects of the isolated compound were assessed in a TNF-α-induced HFLS-RA cell model by evaluating pro-inflammatory cytokine secretion and cell proliferation/metabolic activity. A main flavonoid was identified as formononetin. Formononetin exhibited antioxidant activity and inhibited TNF-α-provoked inflammatory responses in HFLS-RA cells under cell-safe concentrations, as evidenced by reduced secretion of IL-6, IL-8, and IL-1β and suppression of TNF-α-induced hyperproliferation. In conclusion, formononetin is a major flavonoid constituent of L. lucidus var. hirtus, and these findings provide preliminary in vitro cellular evidence that formononetin exerts in vitro anti-inflammatory inhibitory activity on TNF-α-activated human RA fibroblast-like synoviocytes. Further in vivo and mechanistic studies are required to explore its potential for anti-RA-related pharmacological application.