New insights on the anti-inflammatory potential and safety profile of Thymus carnosus and Thymus camphoratus essential oils and their main compounds

Ethnopharmacological relevance Thymus camphoratus and T. carnosus are widely used in Portugal for the treatment of inflammatory-related conditions, such as inflammation of the respiratory tract, being the later also used as an antitussive. Aim of the study Bearing in mind the lack of scientific studies focused on the pharmacological activity of Thymus camphoratus and T. carnosus , this work was designed to validate the anti-inflammatory properties ascribed to these traditional species and concomitantly to unveil both the putative molecular mechanisms behind their bioactivity as well as the safety profile of their essential oils and major compounds. Materials and methods The chemical composition of the essential oils was assessed by gas chromatography (GC) and gas chromatography – mass spectroscopy (GC/MS). The nitric oxide (NO) scavenging potential of the oils was tested using S-nitroso-N-acetyl-D,L-penicillamine (SNAP) as NO donor. The anti-inflammatory potential of the essential oils and their major compounds was evaluated by measuring the nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated macrophages as well as the expression of the pro-inflammatory enzymes, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Importantly, and in an attempt to assess the safety profile of the oils and respective major compounds, their effect on macrophages and hepatocytes viability was also determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Results T. carnosus essential oil was characterized by high amounts of borneol and camphene whereas T. camphoratus oil was rich in 1,8-cineole and borneol. The later presented higher pharmacological activity showing inhibitory effects towards NO production at lower concentrations (0.16 µL/mL) and concomitantly inhibiting the expression of two crucial pro-inflammatory proteins, iNOS and COX-2 (at 0.32 µL/mL). Since no NO scavenging activity was achieved, it is reasonable to conclude that the anti-inflammatory activity of the essential oils occurs upstream of iNOS expression, probably through inhibition of relevant pro-inflammatory signal transduction pathways. Importantly, at bioactive concentrations, the essential oils were devoid of toxicity towards macrophages and hepatocytes. The activity of the isolated compounds was far from that observed for the essential oils, thus suggesting that the anti-inflammatory activity is due to a synergic effect between several compounds in the mixture. Conclusion Overall, the results herein presented sustain and strengthen the anti-inflammatory properties traditionally ascribed to T. carnosus and T. camphoratus. Additionally, the molecular mechanisms associated to their pharmacological activity were highlighted, opening new avenues for the development of effective anti-inflammatory herbal medicinal products.