Investigation into the metabolism of 1,8-cineole in an intestinal cell culture model and acquisition of its immune-modulatory effect via gene expression analysis † Jakob Müller, a * Natalie Gruner, b Isabella Almstätter, a Frauke Kirsch, b Andrea Buettner b,c and Michael W Pfaffl a ABSTRACT: 1,8-Cineole, a common and widely used odorant with antiphlogistic and anti-inflammatory properties, was investigated in this study with regard to potential physiological effects targeting mainly its intestinal effects. Accordingly, the aim of the study was to utilize a combinatory methodological approach to both monitor potential biotransformatory effects on a chemo-analytical basis, as well as physiological and immunological tools to monitor further effects of biofeedback. Reverse transcription quantitative real-time polymerase chain reaction was used to monitor the occurrence of relative expression changes for particular marker genes, following 1,8-cineole treatment. Furthermore, a potential effect of 1,8-cineole on the proliferation and fitness of the intestinal cells using impedance sensing was studied. Generally, our studies showed that the applied model system did neither lead to any significant metabolite formation, nor did the applied dosages result in any major modifications with regard to gene expression. Also, it was shown that cineole had no effect on the intestinal porcine epithelial cells applied in pharmacological or physiological concentrations; neither during the attachment and spreading process nor on confluent cell layers. Only the exposure to high concentrations of cineole (> 1 g/l) affected the cells and led to massive cell detachment. Overall, our studies show that even common higher 1,8-cineole dosages do not seem to lead to any major physiological or aversive response, only until a critical concentration is reached that then directly leads to cell death within the intestinal model. Copyright © 2012 John Wiley & Sons, Ltd. Keywords: hydroxycineole; metabolism; gas chromatography–mass spectrometry; RT-qPCR; ECIS; stable isotope dilution assay Introduction 1,8-Cineole (also named eucalyptol or 1,3,3-trimethyl-2-oxabicyclo [2.2.2]octane) is a potent and characteristic odour substance in diverse plant materials that are also part of the human diet. It is also frequently used for its positive physiological effects in aroma formulations and as a pharmacological agent. [1,2] 1,8-Cineole is the major constituent of oil from leaves of the eucalyptus tree (Eucalyptus sp.), and concentrations may reach 84%. [2] As a result, Eucalyptus sp. is the major natural source of this volatile secondary plant compound. However, other edible plants such as thyme (Thymus sp.) or sage (Salvia sp.) also contain this odorous substance. [3,4] Among other applications 1,8-cineole is used as a therapeutic agent in connection with respiratory diseases. [5,6] Today, different pharmacological preparations based on 1,8-cineole are available (e.g. Soledum W from Klosterfrau Healthcare Group, Köln, Germany; GeloMyrtol W from G. Pohl- Boskamp GmbH & Co, Hohenlockstedt, Germany). They are usually applied orally as enteric coated dragées. In this contained form, the drug traverses the stomach and is eventually released into the small intestine where absorption takes place. The anti-inflammatory, antiphlogistic and anti-infectious capacity of 1,8-cineole is a proven and commonly utilized effect. [2,5,7] Nevertheless, other potential physiological aspects of 1,8-cineole have not been addressed so far. However, when keeping the relatively high dosages of common 1,8-cineole treatments in mind, it is more than justified to look for other possible effects, or at least to exclude the potential occurrence of any negative physiological significance. With regard to the further fate of cineole in the human body after ingestion, some studies demonstrated that 1,8-cineole is metabolized by the cytochrome P450 system in the mammalian liver. [8,9] Also, metabolite profiles in plasma were investigated with regard to their qualitative and quantitative composition. [10] However, the potentially diverse physiological and immunological roles of 1,8-cineole remained largely unclarified. * Correspondence to: Jakob Müller, Physiology Weihenstephan, Technical University Munich, Research Center for Nutrition and Food Science, Weihenstephaner Berg 3, 85350 Freising, Germany. E-mail: jakob. mueller@wzw.tum.de † This article is published in Flavour and Fragrance Journal as Part II of Special Issue: 13 th Weurman Flavour Research Symposium, Zaragoza, Spain, 27 th –30 th September 2011, edited by Vicente Ferreira (University of Zaragoza). a Physiology Weihenstephan, Technical University Munich, Research Center for Nutrition and Food Science, Weihenstephaner Berg 3, 85350 Freising, Germany b Department of Chemistry and Pharmacy – Emil Fischer Center, University of Erlangen–Nuremberg, Schuhstr. 19, 91052 Erlangen, Germany c Fraunhofer Institute for Process Engineering and Packaging (IVV), 85354 Freising, Germany Flavour Fragr. J. 2012, 27, 405–413 Copyright © 2012 John Wiley & Sons, Ltd. Special Issue Paper Received: 2 January 2012, Revised: 3 June 2012, Accepted: 4 June 2012 Published online in Wiley Online Library: 26 September 2012 (wileyonlinelibrary.com) DOI 10.1002/ffj.3109 405