Anti-Inflammatory Effects of Rosmarinus officinalis Essential Oil in Mice

Essential oils are plant secondary metabolites possessing various pharmacological properties, primarily anti-oxidative, antimicrobial or immunomodulative, but they can exhibit toxic and allergic effects as well. The aim of this study was to compare the effects of Rosmarinus officinalis essential oil dietary administration in carrageenan paw oedema and trinitrobenzene sulfonic acid (TNBS) colitis. ICR mice received rosemary essential oil at three concentrations (1 250, 2 500 and 5 000 ppm) in the standard laboratory diet starting two weeks before the experiments. The inflammation of paws induced by carrageenan application was evaluated by measurement of paw swelling, paw weight and myeloperoxidase activity. In the TNBS model the mice were killed by cervical dislocation 3 days after colitis induction and the mortality, changes in the body weight of mice, colon weight : body weight ratio, macroscopical scores and myeloperoxidase activity were analysed. Furthermore, IL-1β and IL-6 cytokine levels in colonic tissue were quantified using ELISA assay. Dietary supplementation with 5 000 ppm of rosemary essential oil initially after 2 h increased but after 24 h suppressed the extent of paw oedema. The same dose in the TNBS model exhibited protective effects on colonic mucosa and significantly decreased macroscopic scores for colonic inflammation. On the other hand, in colon samples from mice fed the diet with 1 250 ppm of rosemary essential oil we detected decreased myeloperoxidase activity and significantly lower levels of IL-6 compared to TNBS control animals. Our results indicate that rosemary essential oil is able to influence several variables of murine experimental inflammatory models depending on the concentration used. We conclude that the anti-inflammatory effects of rosemary essential oil should be interpreted carefully due to its timeand dose-related effects. Inflammation, TNBS colitis, carrageenan paw oedema, rosemary essential oil, cytokines It is known that a large number of plant species contain various bioactive compounds that may have health-beneficial properties, anti-oxidative, anti-inflammatory and antimicrobial effects, and their preventive and therapeutic use is increasing. Various plant products have been already tested in different animal models of inflammation for the development of new preventive and curative remedies. Carrageenan-induced mouse paw oedema has been widely used for analysis of the antiinflammatory effects of plant extracts and essential oils (Maruyama et al. 2006; Fernandes et al. 2007; Juhás et al. 2008a). Experimental colitis in mice and rats induced by the administration of various chemical agents is frequently used to study various compounds as possible therapeutic agents for inflammatory bowel disease (Ikeda et al. 2008). The intrarectal application of trinitrobenzene sulfonic acid (TNBS) produces severe necrotic lesions that heal after several weeks and leave scars and fibrosis as sequelae. Moreover, this model of colitis significantly increased IFN-γ levels in colonic samples only (PérezNavarro et al. 2005). Currently used therapies of inflammatory bowel disease seem to be suboptimal and consequently different complementary and alternative medicines are used for symptom relief and improved quality of life. Accordingly, many of these alternative therapies are able to modulate the immune system and disrupt the proinflammatory cascade through a variety of mechanisms, including antioxidant effects, alterations in ACTA VET. BRNO 2009, 78: 121–127; doi:10.2754/avb200978010121 Address for correspondence: Juraj Koppel, DVM, PhD Institute of Animal Physiology, Slovak Academy of Sciences Šoltésovej 4-6 040 01 Košice Slovak Republic Phone: +421 55 7287841 Fax: +421 55 7287842 E-mail: [email protected] http://www.vfu.cz/acta-vet/actavet.htm cell signalling (in particular the NF-κB pathway), cytokines, proinflammatory mediators, and disruption of bacterial flora (Clarke and Mullin 2008). These alternative treatments include herbal therapy consisting of anti-oxidant, anti-inflammatory and antimicrobial plants administration, too. We have described very recently that thyme essential oil in the diet was able to attenuate murine TNBS-induced colitis depending on the concentration used (Juhás et al. 2008a). Rosmarinus officinalis is known as a common herb and household plant broadly used all around the world for different medicinal purposes, being a component of various established anti-inflammatory plant drug preparations, and having a long tradition of use for treating headaches, colds and colic, as well as other diseases (Darshan and Doreswamy 2004). Anti-oxidant properties of rosemary and their compounds have been confirmed recently (Cheung and Tai 2007; Atsumi and Tonosaki 2007); furthermore, rosemary extracts and their triterpenes (ursolic acid, oleanolic acid and micromeric acid) have been shown to exert anti-inflammatory activity in vivo (Altinier et al. 2007). Up to now only a few studies have analysed the effects of rosemary or its essential oil on the gastrointestinal apparatus (Galisteo et al. 2000). Our aim therefore was to examine whether the dietary addition of rosemary essential oil could have positive effects in experimental intestinal inflammation induced by TNBS administration in mice. Materials and Methods Animals and treatment Weight-matched ICR mice (5–6-week-old males from the Institute’s breeding facility, weighing 28–32 g) were randomized into groups. The organization of the experiment, the investigations conducted, and the related documentation complied fully with legislative regulations governing the protection of experimental animals in the Slovak Republic. All animal experimentation was reviewed and approved by the Ethics Committee of the Institute of Animal Physiology. Rosmarinus officinalis essential oil (Ph. Eur. 4) purchased from Calendula, a.s. (Nová Ľubovňa, Slovakia, lot 5-014-009-12-06, containing approx. 25% cineole, 19% α-pinene, 19% camphor, 17% p-cymene, 9% camphene, 5% -pinene and 2% borneol) was added to powdery commercial rodent diet (Diet for laboratory mice and rats SPF, M1; František Machal, Ricmanice, Czech Republic) in 1% edible soy oil (Brölio, Germany) at the following concentrations: 5 000 ppm (wt/wt) group A; 2 500 ppm (wt/wt) group B; 1 250 ppm (wt/wt) group C. The diet for control and sham groups was prepared similarly using only 1% edible soy oil. The diets were fed ad libitum during the experimental period starting 14 days before administration of TNBS enemas. The mice of experimental group D were treated with dexamethasone (DEXAMED, Medochemie Ltd., Limassol, Cyprus) at a dose of 3 mg/kg b.w. s.c. 2 h before carrageenan application (paw oedema model) or 0.25 mg/kg b.w. s.c. starting 2 h before TNBS instillation, and then every 24 h until the sacrifice of the animals. Dexamethasone has anti-inflammatory effects and attenuates the enhanced neutrophil infiltration in many animal inflammatory models including carrageenan paw oedema and TNBS colitis. Carrageenan paw oedema Male ICR were anaesthetized with the mixture [ketamine 5% (42.5%) / xylasine 2% (7.5%) / NaCl 0.9% (50%)] at 60 μl/20 g body weight i.p. Each group of animals received subplantar administration of 50 μl of saline to the left paw or 50 μl of carrageenan 1% (w/v) (SIGMA, Steinheim, Germany) in saline to the right paw. The volume was measured using a Mitutoyo thickness gauge (Mitutoyo, No. 7313, Japan) immediately before subplantar injection, and 2, 4, 24 h thereafter. The increase in paw volume was calculated as the difference between the right paw volume (carrageenan) and the left paw volume (saline) measured at each time point. The mice were killed by cervical dislocation after the last time point (24 h) and the carrageenan paws were rapidly amputated at the tarsocrural joint and weighed on an analytical balance. Induction of TNBS colitis Mice were anaesthetized with the mixture [ketamine 5% (42.5%) / xylasine 2% (7.5%) / NaCl 0.9% (50%)] at 60 μl/20 g body weight i.p. and colitis was induced by intrarectal administration of 120 mg/kg of the hapten reagent TNBS (Fluka, Steinheim, Germany) in 50% ethanol, and they were then kept in a vertical head-down position for 30 s. The sham group received 50% ethanol alone through the same technique. The total injection volume was 40 μl. Development of colitis was assessed daily by the measurement of body weight. The mortality rate was observed during this study. Mice were killed by cervical dislocation 3 days after TNBS administration. Afterwards the colons were removed, opened longitudinally and cleared of faecal material with a gentle spray of 0.9% saline solution. The extent of mucosal damage was assessed using the colon macroscopic scoring system (Bukovská et al. 2007, adapted from Wallace et al. 1989). 122