Comparative Efficacy of Fermented and Unfermented Ethanolic Rooibos Extracts in Mitigating LPS-Induced Intestinal Inflammation and Barrier Dysfunction

Rooibos (Aspalathus linearis) is a rich source of bioactive polyphenols; however, most biological studies have focused on traditional aqueous tea extracts, despite extraction solvent and enrichment procedures substantially influencing phytochemical composition and biological activity. This study investigated whether chemically distinct enriched ethanolic extracts prepared from unfermented and fermented rooibos differentially modulate intestinal inflammation and barrier integrity. Following ethanol extraction and Amberlite XAD-4 enrichment, the extracts were characterized for soluble solids, flavonoid composition and antioxidant capacity before evaluation in an Escherichia coli lipopolysaccharide (10 μg/mL)-stimulated IPEC-J2 intestinal epithelial cell model. Anti-inflammatory activity was assessed by determining cell viability, caspase activity, IL-8 protein production, and gene expression of IL-8, IL-6 and TLR4, while barrier integrity was evaluated using transepithelial electrical resistance (TEER), ZO-1 immunofluorescence and tight junction gene expression. Ethanol extraction and enrichment generated extracts with markedly different phytochemical profiles. The enriched unfermented extract contained substantially higher soluble solids (77.72 g/100 g) and aspalathin (60 g/100 g soluble solids) than the fermented extract (53.10 g/100 g and 9.46 g/100 g, respectively), resulting in significantly greater antioxidant activity. Despite these compositional differences, both extracts attenuated LPS-induced inflammation by reducing IL-8 protein production and downregulating IL-8, IL-6 and TLR4 gene expression. However, distinct biological activities were observed. The polyphenol-rich unfermented extract provided superior protection of epithelial barrier integrity, demonstrated by increased TEER, enhanced ZO-1 localisation and upregulation of tight junction markers, whereas the fermented extract exhibited pronounced anti-inflammatory activity at lower concentrations despite its lower polyphenol content. These findings demonstrate that post-harvest processing and extraction technique determine the phytochemical composition of rooibos and consequently influence its dose-dependent biological activity. Tailoring extraction strategies may therefore enable the development of rooibos extracts towards certain product formulations with targeted anti-inflammatory or barrier-protective properties.