Protection against LPS-induced cartilage inflammation and degradation provided by a biological extract of Mentha spicata

BACKGROUND: A variety of mint [Mentha spicata] has been bred which over-expresses Rosmarinic acid (RA) by approximately 20-fold. RA has demonstrated significant anti-inflammatory activity in vitro and in small rodents; thus it was hypothesized that this plant would demonstrate significant anti-inflammatory activity in vitro. The objectives of this study were: a) to develop an in vitro extraction procedure which mimics digestion and hepatic metabolism, b) to compare anti-inflammatory properties of High-Rosmarinic-Acid Mentha spicata (HRAM) with wild-type control M. spicata (CM), and c) to quantify the relative contributions of RA and three of its hepatic metabolites [ferulic acid (FA), caffeic acid (CA), coumaric acid (CO)] to anti-inflammatory activity of HRAM. METHODS: HRAM and CM were incubated in simulated gastric and intestinal fluid, liver microsomes (from male rat) and NADPH. Concentrations of RA, CA, CO, and FA in simulated digest of HRAM (HRAM(sim)) and CM (CM(sim)) were determined (HPLC) and compared with concentrations in aqueous extracts of HRAM and CM. Cartilage explants (porcine) were cultured with LPS (0 or 3 μg/mL) and test article [HRAM(sim )(0, 8, 40, 80, 240, or 400 μg/mL), or CM(sim )(0, 1, 5 or 10 mg/mL), or RA (0.640 μg/mL), or CA (0.384 μg/mL), or CO (0.057 μg/mL) or FA (0.038 μg/mL)] for 96 h. Media samples were analyzed for prostaglandin E(2 )(PGE(2)), interleukin 1β (IL-1), glycosaminoglycan (GAG), nitric oxide (NO) and cell viability (differential live-dead cell staining). RESULTS: RA concentration of HRAM(sim )and CM(sim )was 49.3 and 0.4 μg/mL, respectively. CA, FA and CO were identified in HRAM(sim )but not in aqueous extract of HRAM. HRAM(sim )(≥ 8 μg/mL) inhibited LPS-induced PGE(2 )and NO; HRAM(sim )(≥ 80 μg/mL) inhibited LPS-induced GAG release. RA inhibited LPS-induced GAG release. No anti-inflammatory or chondroprotective effects of RA metabolites on cartilage explants were identified. CONCLUSIONS: Our biological extraction procedure produces a substance which is similar in composition to post-hepatic products. HRAM(sim )is an effective inhibitor of LPS-induced inflammation in cartilage explants, and effects are primarily independent of RA. Further research is needed to identify bioactive phytochemical(s) in HRAM(sim).