Effects of 1,8-cineole (eucalyptol) on the activity of human bronchial tissue

1,8-cineole (eucalyptol) is used for the treatment of bronchial complaints, sinusitis and colds. Experiments have previously shown that 1,8-cineole, a monoterpene (C-10), has a very pronounced spasmolytic effect on smooth muscle fibre. In nearly all clinical applications with 1,8-cineole, especially when used in conjunction with allergic symptoms, histamine receptors prove to be crucial for treatment (use of histamine inhibitors). Results: The desired 1,8-cineole effects are attained through: 1. specifically blocking H1 histamine receptors, without influencing ACh receptors 2. inhibiting the contractile activity of human bronchial smooth muscle by activating H2 histamine receptors. The ultimate goal of this study was to address the bronchodilatory effect of this compound, using human airway smooth muscle in order to demonstrate a possible role for 1,8-cineole in airway diseases.


Introduction
For many years, medications containing 1,8cineole (eucalyptol) have been used for the treatment of bronchial complaints, sinusitis and colds. It has been previously shown that 1,8cineole, a monoterpene (C-10) [1], has a very pronounced spasmolytic effect on smooth muscle fibres (SMF), similar to that of papaverine [2]. 1,8-cineole was found to have agonistic effects on the 1 and 2 adrenergic receptors [3]. These effects can be registered at low concentrations of up to 3 x10 -7 M to 2 x 10 - 5  Page: 13 www.raftpubs.com well-known spasmolytic effect appears. At concentrations above 4 x 10 -4 M 1,8-cineole, the effect of 10 -5 M acetylcholine is 100% suppressed. The results of the interactions between 1,8-cineole and histamine indicate that 1,8-cineole is a reversible antagonist for histamine receptors. Increasing the "concentration" of 1,8-cineole by 16-fold (from 0.005 up to 0.080 l) increases this inhibition from approx. 25% to approx. 66% [4]. However, the various smooth muscles in the body are characterised by different types and number of receptors. The aim of this manuscript is to describe the effect of 1,8-cineole on the contractile activity of human bronchial smooth muscles.

Materials and Methods
In in vitro experiments on smooth muscle fibre of bronchial muscle, the influence of histamine under normal conditions and using different concentrations of 1,8-cineole on the spontaneous contractile activity (SCA) of SMF was investigated.
2.1 Measurement of the spontaneous contractile activity of smooth muscle fibre of segmental bronchi and method of preparation The measurements were performed according to the standardised Golenhofen method [5]. The smooth muscle fibre used in the experiments was taken from human segmental bronchi.

Statistics
To take into account the specific variations and alterations of the SCA preparation's measured values, the concentration-response curves in each case of N = 10 individual experiments, always measured the excitation in % of maximal contractile activity of smooth muscle tissue when exposed to 10 -5 M acetylcholine (ACh). The changes of the smooth muscle fibre (SMF) concerning SCA with various substances are given in Newtons (N). The processing of experimental data was performed using the Statistica 4.5 (StatSoft, Inc. Microsoft, USA) program. For comparison between two groups, the t-Test (student) for unpaired samples was implemented. For comparisons between three or more groups, variance analysis (ANOVA) was used. The statistical comparisons were performed at the 5% significance level. The results are expressed as mean ± standard deviation. In each case n = 7 measurements per experiment were performed.

Discussion
1,8-cineole is a monoterpene known to be a component of various essential oils, e.g. of the genus Eucalyptus, Salvia, Rosmarinus, but is mainly isolated from Eucalyptus species which produce essential oil rich in cineole. This saturated terpene has a number of medicinally useful anti-inflammatory, anti-oxidative and antimicrobial effects, as recently presented in an overview by Juergens [6]. An experimental study provides useful data suggesting that 1,8-cineole is a specific COX-2 blocker [7]. 1,8-cineole is used as an active ingredient in medicinal products and can be inhaled, topically applied or be taken orally. After resorption of 1,8-cineole in the small intestine part of it is eliminated unchanged by exhalation [8]. In the intestinal as well as the bronchial tract, 1,8-cineole also comes into contact with smooth muscle fibre. Investigations of the effects of 1,8-cineole on SMF, whether receptor-specific or receptor-independent, are expected to contribute to understanding of the clinical efficacy of 1,8-cineole, e.g. in inflammatory bronchial diseases [9]. Our results show that the mechanisms of this inhibition are due to an effect specific to the human bronchial system. Biologically active substances can influence the contractile activity of smooth muscle in various ways. Firstly, the contractile activity of smooth muscle can be influenced by an effect on the receptors present on smooth muscle cells, e.g. through the effects of histamine, dopamine, acetylcholine, other mediators or with synthetic substances. For influences at the receptor level, the effects on the contractile activity of smooth muscle typically only occur after several seconds. The second notable aspect of these effects is that demonstrable changes in contractile activity are still observed at concentrations of 10 -7 -10 -9 M. Very often, these are reversible.
Secondly, biologically active substances have effects on different enzyme systems (e.g. phosphodiesterases, adenylate cyclases, cyclooxygenases). For such substances, the effects typically occur only after several Page: 20 www.raftpubs.com minutes and at concentrations above 10 -6 M. Independent of other properties of a substance (spasmolytic effects etc.), these time and concentration intervals allow the effects of biologically active substances to be analysed at the receptor level. This in particular applies to substances exhibiting multiple effects on smooth muscle, such as, for example, some monoterpenes (1,8- It was also demonstrated that 1,8-cineole, at a concentration of 3 x 10 -6 M, has agonistic effects on α1 und α2 adrenoreceptors [3]. Activation of α1 and α2 adrenoreceptors, which also occurs with other monoterpenes, e.g. with thymol, is observed at very high concentrations [10]. This is the reason for the existence of contradictory reports on the effect of 1,8cineole on the bronchi. At a low doses, the stimulating effects of 1,8-cineole dominate through the activation of the α1 and α2 adrenoreceptors. Additional intensification of the bronchial spasmolytic properties results as a consequence of the mentioned effects. At somewhat higher concentrations, an equilibrium occurs between the stimulating and suppressive effects on the contractile activity of the bronchi. A pronounced spasmolytic effect on the bronchi is recorded at higher concentrations. At sufficiently high concentrations of 1,8cineole (approx. 10 -5 M in vitro), contraction is blocked due to the stimulation of α1 and α2 adrenoreceptors by its spasmolytic effect, as there are insufficient free Ca 2+ ions available. The spasmolytic effect of 1,8-cineole cannot influence the effects on the H1 and H2 histamine receptors and the blocking of cyclooxygenase. As a result, the desired 1,8-cineole effects are attained: 1. A specific blocker of H1 histamine receptors, with no effect on ACh receptors.
2. Inhibition of the contractile activity of human bronchial smooth muscle through activation of H2 histamine receptors.
These results are in line with former investigations indicating the spasmolytic effect of 1,8-cineole on SMF in our results [3]. A study by Wagner [11] showed, however, that terpenes, when administered to patients with bronchial asthma, resulted in paradoxical bronchoconstriction reactions. We believe that these contradictory reports are based on the special properties of terpenes: Monoterpenes are known to have both specific and nonspecific effects on smooth muscle fibre, dependent on the dose. Some monoterpenes, such as 1,8-cineole and thymol [10], at concentrations of up to 5 x 10 -7 M, lead to constricting effects on almost all smooth muscle fibre. These effects are due to the agonistic effects on α 1 and α 2 adrenoreceptors. However, they can only be observed up to an administered dose of a concentration of 5 x 10 -7 M. Exceeding this amount, the pronounced spasmolytic effect of cineole appears, which no longer allows the registration of specific effects. Low concentrations of 1,8-cineole allow observations of bronchoconstriction. Here, we recorded the stimulating effects of 1,8-cineole on smooth muscle fibre (due to the excitation effects on H 1 receptors). At higher concentrations very pronounced spasmolytic effects can be observed and, of course, inhibition of H 1 receptor activity. The