Optimized extraction of polyphenols from rooibos tea (Aspalathus linearis) and their biological activities

Fermented (red) rooibos tea (Aspalathus linearis) has been widely consumed for its well-known health-promoting effects and represents an important dietary source of natural polyphenols. Yet, compared with unfermented (green) rooibos tea, fermented rooibos tea has received limited investigation, particularly regarding the optimization of its aqueous extraction and the systematic evaluation of its chemical composition and bioactivities. This study aimed to optimize polyphenol extraction from commercial red rooibos tea using response surface methodology (RSM), followed by assessment of the antioxidant, antibacterial, and anticancer activities of the optimized extract. In addition, the phenolic profile of the extract was characterized using ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS). The solid-to-liquid extraction using a water bath was optimized for extraction temperature and time factors. The extract was evaluated for total phenolic content (TPC) using the Folin-Ciocalteu assay, and for antioxidant activity using the DPPH (2,2-diphenyl-picrylhydrazyl) assay. RSM analysis indicated that temperature was the dominant factor influencing both TPC and DPPH activity, with time showing a secondary temperature-dependent effect. At the optimized conditions (90 °C, 10 min), the optimum rooibos tea extract (RTE) showed a TPC of 49.94 mg GAE/g DM and an antioxidant activity of 101.90 mg TE/g DM. UHPLC-QTOF-MS analysis identified 43 metabolites in the RTE, mainly isoorientin, rutin, aesculetin, and aspalathin among others. The RTE produced inhibition zones of 12 mm against Staphylococcus aureus and 10 mm against Pseudomonas aeruginosa in the disc diffusion assay, while no inhibition zones were observed for Bacillus cereus, Listeria monocytogenes, Escherichia coli, or Salmonella Typhimurium. Broth microdilution assays yielded Minimum Inhibitory Concentration/Minimum Bactericidal Concentration (MIC/MBC) values of 25/25 mg/mL for S. aureus, 25/50 mg/mL for L. monocytogenes, 50/50 mg/mL for P. aeruginosa, and 50/100 mg/mL for E. coli, whereas B. cereus and S. typhimurium showed resistance to RTE under the tested conditions. For the anticancer activity, evaluated using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, the extract did not achieve 50% inhibition across the concentrations tested (50-700 μg/mL) against H460, HT-29, and Caco-2 cancer cell lines. This study is the first to integrate RSM-based optimization of aqueous red rooibos extraction with chromatographic profiling and in vitro biological assays. Future studies may compare red and green rooibos tea, and explore product variability, in vivo relevance, and alternative processing strategies.