Pharmacological screening of essential oils for identification of NF-κB and iNOS inhibitors in a human intestinal epithelial cell line

I Ferreira1, AT Rufino1,2, MC Lopes1, L Salgueiro3, C Cavaleiro3, AF Mendes1,2. 1Center for Neuroscience and Cell Biology, 3004-517 Coimbra, Portugal, 2University of Coimbra, Faculty of Pharmacy, 3000-548 Coimbra, Portugal, 3University of Coimbra, Lab of Pharmacognosy, Faculty of Pharmacy/CEF, 3000-548 Coimbra, Portugal

Purpose: Inflammatory bowel disease (IBD) is a complex and devastating condition affecting millions of people, for which no effective therapies are yet available. One promising target is Nuclear Factor-kappaB (NF-κB), which contributes to IBD by promoting the development and perpetuation of chronic inflammation, namely through the expression of the inducible nitric oxide synthase (iNOS). Essential oils (EOs) are complex mixtures of lipophilic low molecular weight (130 to 300 dalton) plant secondary metabolites, encompassing a huge diversity of terpenoids and/or phenylpropanoids. Due to favourable pharmacokinetic properties and chemical diversity, EOs are promising samples to screen for pharmacologically active compounds. This study aimed at screening a set of five essential oils and one pure compound, α-pinene, previously found to be effective in human chondrocytes (1), for the ability to inhibit NF-κB activation and iNOS expression induced by 1000 U/mL IFN-γ, 10 ng/mL IL-1β and 10 ng/mL TNF-α, a mixture of pro-inflammatory cytokines (CytMix) known to be involved in the pathogenesis of IBD. The EOs of Thapsia villosa, Otanthus maritimus, Lavandula luisieri, Laserpitium eliasii and Annona muricata were selected because together, they represent the major chemical families usually found in EOs and/or because of their etnopharmacological indication as anti-inflammatory agents.

Methods: The human colorectal adenocarcinoma cell line, C2Bbe1, a more differentiated clone of the parent cell line, Caco-2, was cultured in DMEM high glucose with 10% FBS and pre-treated with each EO for 30min before addition of the CytMix. The first screen consisted in evaluating the ability of each EO to decrease the CytMix-induced iNOS protein levels. The active EOs were then evaluated for their ability to inhibit NF-κB activation. The MTT reduction assay was used to rule out cytotoxic effects. Protein levels of iNOS, total and phosphorylated IκB-α were assessed after treatment with CytMix for 18h, 30min or 5min, respectively, by western blot and normalized to the respective actin band used as a loading control. The results (n≥4) relative to the CytMix were expressed as the mean±SD and considered significant for P<0.05.

Results: Three EOs were effective in reducing CytMix-induced iNOS expression, although with different potencies. The greatest inhibition was achieved with 0.2 mg/mL of L. luisieri EO, which reduced iNOS levels to 19.0±11.6% (P<0.0001) of those found in cells treated with the CytMix alone (1.355±1.786). Although much less efficiently, the same concentration of the oils from L. eliasii (54.7±17.4%, P<0.05) and O. maritimus (54.8±15.2%, P<0.001) also reduced iNOS levels. Unlike in human chondrocytes, α-pinene did not affect CytMix-induced iNOS expression. L. luisieri (0.2 mg/mL) also significantly reduced IκB-α phosphorylation to 37.7 ± 8.0% (P<0.0001) of the CytMix-induced response (0.421±0.195). Phosphorylated IκB-α was undetectable in untreated cells. Total IκB-α levels increased from 11.7±3.9 % (0.251±0.196; P<0.0001 vs control) in CytMix-treated cells to 33.4±14.7% (P<0.0001 vs control, P<0.01 vs CytMix) in L. luisieri-treated cells relative to the control (2.276 ±1.732), indicating that the EO effectively decreased NF-κB activation.

Conclusion: The EO of L. luisieri is the most promising as an inhibitor of NF-κB activation and iNOS expression in C2Bbe1 cells. Additional studies are underway to isolate and identify the active compound(s) and further characterize the mechanism(s) involved in their anti-inflammatory action.

1. Neves A. et al. Planta Med. 2010; 76:303-8