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

Pearson W, Fletcher RS, Kott LS, Hurtig MB - BMC Complement Altern Med (2010)

Bottom Line: No anti-inflammatory or chondroprotective effects of RA metabolites on cartilage explants were identified.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Dept Plant Agriculture, University of Guelph, Ontario, Canada. wpearson@ovc.uoguelph.ca

ABSTRACT

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 (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 microg/mL) and test article [HRAMsim (0, 8, 40, 80, 240, or 400 microg/mL), or CMsim (0, 1, 5 or 10 mg/mL), or RA (0.640 microg/mL), or CA (0.384 microg/mL), or CO (0.057 microg/mL) or FA (0.038 microg/mL)] for 96 h. Media samples were analyzed for prostaglandin E2 (PGE2), interleukin 1beta (IL-1), glycosaminoglycan (GAG), nitric oxide (NO) and cell viability (differential live-dead cell staining).

Results: RA concentration of HRAMsim and CMsim was 49.3 and 0.4 microg/mL, respectively. CA, FA and CO were identified in HRAMsim but not in aqueous extract of HRAM. HRAMsim (> or = 8 microg/mL) inhibited LPS-induced PGE2 and NO; HRAMsim (> or = 80 microg/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. 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.

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HPLC chromatogram of aqueous extract [A] and biological extract B] of High-Rosmarinic Acid Mint [HRAM]. [A] The major peak represents rosmarinic acid [RA] at a concentration of 130 ug/mL. [B] Retention times of compounds of interest: Caffeic acid = 6.00 min; Ferulic acid = 10.52 min; m-Coumaric acid = 14.10 min; Rosmarinic acid = 20.50 min.
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Figure 1: HPLC chromatogram of aqueous extract [A] and biological extract B] of High-Rosmarinic Acid Mint [HRAM]. [A] The major peak represents rosmarinic acid [RA] at a concentration of 130 ug/mL. [B] Retention times of compounds of interest: Caffeic acid = 6.00 min; Ferulic acid = 10.52 min; m-Coumaric acid = 14.10 min; Rosmarinic acid = 20.50 min.

Mentions: Chromatogram of HRAM aqueous extract and HRAM simulated digest is provided in Figure 1A and 1B respectively. Concentrations of RA and its primary hepatic metabolites identified in HRAMsim, CMsim, and aqueous extracts of HRAM and CM are reported in Table 1.


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

Pearson W, Fletcher RS, Kott LS, Hurtig MB - BMC Complement Altern Med (2010)

HPLC chromatogram of aqueous extract [A] and biological extract B] of High-Rosmarinic Acid Mint [HRAM]. [A] The major peak represents rosmarinic acid [RA] at a concentration of 130 ug/mL. [B] Retention times of compounds of interest: Caffeic acid = 6.00 min; Ferulic acid = 10.52 min; m-Coumaric acid = 14.10 min; Rosmarinic acid = 20.50 min.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2874512&req=5

Figure 1: HPLC chromatogram of aqueous extract [A] and biological extract B] of High-Rosmarinic Acid Mint [HRAM]. [A] The major peak represents rosmarinic acid [RA] at a concentration of 130 ug/mL. [B] Retention times of compounds of interest: Caffeic acid = 6.00 min; Ferulic acid = 10.52 min; m-Coumaric acid = 14.10 min; Rosmarinic acid = 20.50 min.
Mentions: Chromatogram of HRAM aqueous extract and HRAM simulated digest is provided in Figure 1A and 1B respectively. Concentrations of RA and its primary hepatic metabolites identified in HRAMsim, CMsim, and aqueous extracts of HRAM and CM are reported in Table 1.

Bottom Line: No anti-inflammatory or chondroprotective effects of RA metabolites on cartilage explants were identified.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Dept Plant Agriculture, University of Guelph, Ontario, Canada. wpearson@ovc.uoguelph.ca

ABSTRACT

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 (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 microg/mL) and test article [HRAMsim (0, 8, 40, 80, 240, or 400 microg/mL), or CMsim (0, 1, 5 or 10 mg/mL), or RA (0.640 microg/mL), or CA (0.384 microg/mL), or CO (0.057 microg/mL) or FA (0.038 microg/mL)] for 96 h. Media samples were analyzed for prostaglandin E2 (PGE2), interleukin 1beta (IL-1), glycosaminoglycan (GAG), nitric oxide (NO) and cell viability (differential live-dead cell staining).

Results: RA concentration of HRAMsim and CMsim was 49.3 and 0.4 microg/mL, respectively. CA, FA and CO were identified in HRAMsim but not in aqueous extract of HRAM. HRAMsim (> or = 8 microg/mL) inhibited LPS-induced PGE2 and NO; HRAMsim (> or = 80 microg/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. 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.

Show MeSH