Bioactivity-guided isolation of anti-inflammatory compounds from the herbs constituting a traditional Chinese medicine formulation used for the treatment of rheumatoid arthritis

• A screening platform for testing plant extracts against key RA inflammatory pathways (NF-κB, NFAT, STAT3, STAT5), prostaglandin production, and cytokine release was developed. • A comprehensive extraction method encompassing a range of solvents and fractionation methods allowed for the identification of the most potent chemical constituents from a TCM formulation. • A. sinensis, E. sinica, C. cassia, G. uralensis , and A. lancea showed diverse anti-inflammatory profiles . Milder anti-inflammatory effects were shown by C. lacryma-jobi, P. lactiflora , and A. membranaceus. • 39 compounds showing diverse anti-inflammatory activities encompassing 10 structural classes were isolated, including two new compounds from A. lancea. • The identified anti-inflammatory compounds offer mechanistic insights into bioactive constituents present in a clinically used TCM formulation for RA. Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that poses a significant socio-economic burden. Traditional Chinese medicine (TCM) herbal formulations, with their long history of clinical use, represent a promising source for novel anti-rheumatic drugs. However, the complexity of RA pathogenesis and the chemical diversity of TCM formulations present challenges in understanding compound-target interactions. This study aimed to identify the anti-inflammatory constituents from the individual plant species included in the clinically used TCM formulation (“Formulation A”), and to characterize their effects on key inflammatory pathways. Formulation A is currently undergoing a double-blind, randomized clinical trial. A key innovation of this work is the integration of a phenotypic screening strategy, bridging the gap between typical single-species natural product studies and formulation-level decoction studies. A custom panel of phenotypic in vitro assays reflecting key inflammatory pathways relevant to RA (NF-ĸB, NFAT, STAT3, and STAT5) was developed. A suite of plant extracts and fractions was generated, then screened in the above assays. Effects on cytokine production in primary human B cells (IL-6, TNF-α, and GM-CSF) and prostaglandin production (PGD 2 , PGE 2 , PGF 2α and TxB 2 ) in a fibroblast cell line were also evaluated. Plant species within Formulation A displayed a spectrum of biological activities, from minor to broad and more targeted effects. The strongest anti-inflammatory activities were observed in nonpolar extracts and fractions, reflecting the utility of organic solvent extraction for accessing pharmacologically potent constituents that may be underrepresented in traditional water-based decoctions. Two new compounds from Atractylodes lancea were identified, alongside 37 known compounds from other species exhibiting both new and previously known anti-inflammatory activities. These findings also highlight specific molecular effects of the formulation’s components on inflammatory signaling pathways and mediators. This study provides mechanistic insights into the anti-inflammatory effects of plant constituents present in Formulation A, based on the systematic analysis of each individual plant species. Moreover, it establishes a robust phenotypic screening platform for the systematic discovery of new anti-rheumatic agents from natural sources.