Bone Abstracts

Rooibos (Aspalathus linearis) tea is a refreshing and caffeine-free tea indigenous to the Western Cape regions of South Africa. This tea, which is rich in polyphenols and antioxidants, has shown many beneficial effects in health, however, its anti-osteoclastogenic potential remains unexplored. In this study, the in vitro effects of an aqueous extract of fermented rooibos tea were examined on osteoclast formation and bone resorption in RAW264.7 murine macrophages. RAW264.7 macrophages were seeded at 15,000 cells/cm² in the presence of sterile distilled water (vehicle control) or tea extract (62.5–500 μg/ml). Cell viability was determined by alamar blue assay after 48 h exposure. Osteoclastogenesis was stimulated by the addition of RANKL (15 ng/ml) and evaluated after 5 days by staining for the enzyme tartrate-resistant acid phosphatase (TRAP) and quantification of TRAP-positive multinucleated cells. Actin ring formation was determined by fluorescent stain using a phalloidin conjugate. Bone resorption assays were conducted on osteoassay plates coated with inorganic synthetic bone surface after washing off the cells to visualize resorption pits. The expression of key osteoclast markers was determined via real-time PCR. NF-κB activation was determined by secreted alkaline phosphatase (SEAP) assay after stably transfecting cells with a NF-κB inducible SEAP reporter plasmid. Three independent experiments were conducted in triplicate for each test. We found that rooibos tea extract at 250–500 μg/ml significantly (P<0.05) inhibited osteoclast formation and TRAP activity, which was accompanied by reduced bone resorption and disruption of characteristic cytoskeletal elements of mature osteoclasts, without cytotoxicity. Rooibos tea extract decreased expression of key osteoclast specific genes, matrix metalloproteinase-9, TRAP and cathepsin K. Furthermore, the tea extract inhibited the activation of the intracellular signalling marker, NF-κB, at increasing concentrations after 48 h. This study demonstrates for the first time that rooibos tea may have potential anti-osteoclastogenic effects.