Abstract
BackgroundA 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.MethodsHRAM 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 (HRAMsim) and CM (CMsim) 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 [HRAMsim (0, 8, 40, 80, 240, or 400 μg/mL), or CMsim (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 E2 (PGE2), interleukin 1β (IL-1), glycosaminoglycan (GAG), nitric oxide (NO) and cell viability (differential live-dead cell staining).ResultsRA concentration of HRAMsim and CMsim was 49.3 and 0.4 μg/mL, respectively. CA, FA and CO were identified in HRAMsim but not in aqueous extract of HRAM. HRAMsim (≥ 8 μg/mL) inhibited LPS-induced PGE2 and NO; HRAMsim (≥ 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.ConclusionsOur biological extraction procedure produces a substance which is similar in composition to post-hepatic products. HRAMsim 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 HRAMsim.
Highlights
A variety of mint [Mentha spicata] has been bred which over-expresses Rosmarinic acid (RA) by approximately 20-fold
HPLC analysis Chromatogram of HRAM aqueous extract and HRAM simulated digest is provided in Figure 1A and 1B respectively
prostaglandin E2 (PGE2) LPS significantly increased PGE2 in explants which were not conditioned with HRAMsim (Figure 2A)
Summary
A variety of mint [Mentha spicata] has been bred which over-expresses Rosmarinic acid (RA) by approximately 20-fold. Like pure RA, mint oil inhibits the inflammatory consequences of LPS, including inhibition of interleukin (IL-1), prostaglandin E2 (PGE2), leukotriene B4 (LTB4) production by LPS-stimulated human monocytes [9]. As these biological actions are considered to be related to the RA content of the plant, considerable effort has been invested in developing strategies to upregulate biosynthesis of RA by genetically modified (GMO) plant tissues [10,11]. There remains a commercial opportunity for agronomic selection of plants with naturally robust biosynthesis of RA for the nutraceutical and biotechnology markets
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