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Investigations into the specific effects of rosemary oil at the receptor level.

ABSTRACT

Rosemary oil is used frequently in phytotherapy. The objective of the present study was to investigate the extent to which rosemary oil shows other effects on the smooth muscles than the familiar spasmolytic effects. The effects of rosemary oil on the spontaneous contractile activity were investigated in in vitro experiments with circular smooth-muscle strips of the guinea pig stomach. Rosemary oil was found to have agonistic effects on the [[alpha].sub.1] and [[alpha].sub.2] adrenergic receptors. These effects can be registered at concentrations up to 25 [micro]l/l of rosemary oil. At higher concentrations the spasmolytic effect described in other reports could be detected. At concentrations above 100 [micro]l/l rosemary oil, the effect of [10.sup.-5]M ACH is completely suppressed. The results permit the assumption that, besides the spasmolytic effects investigated to date, owing to its specific effects on the [[alpha].sub.2] adrenergic receptors of the nerve cells, rosemary oil brings about an additional improvement of local blood circulation and alleviates pain.

Keywords: Smooth muscle fibres Rosemary oil [[alpha].sub.1]-[[alpha].sub.2] adrenergic receptors Papaverine Pain Clonidine

Introduction

Rosemary oil is an essential oil produced by means of steam distillate of the above-ground blossoming parts of rosmarinus officinalis L, which can be applied in practice on its own or combined with other essential oils. Rosemary oil contains virtually only substances from the group of the terpenes. The areas of application for rosemary oils are considered to be for dyspeptic complaints (4-6 g drug, to be taken internally), as an adjuvant for rheumatic diseases (external use, bath supplement, spirit, ointment), for circulatory complaints (external use), additionally to prompt wound healing, as a mild antiseptic and for use on the biliary tract and the small intestine because of its spasmolytic effects (Schilcher et al. 2007). The antibacterial and spasmolytic effect of rosemary oil have been described in the literature (Santoyo et al. 2005; Cabo et al. 1986; Taddei et al 1988; Imaseki and Kitabatake 1962; Wagner et al. 1986).

However, to date there have been no investigations to establish if rosemary oil has specific effects, for example on receptors, comparable with other extracts, we have already investigated (Beer et al. 2007). Investigations of this nature are impeded by the fact that rosemary oil has spasmolytic, Papaverine-like effects (Forster et al. 1980). At the same time it is also known that the specific effects normally occur when the concentrations of the agents are significantly lower. For example, one obtains a concentration interval of the agent between effectiveness of the specific and non-specific effects. This concentration interval is generally around 2 powers of ten. The objective of the present paper is to investigate the extent to which rosemary oil - total extract - also proves to have other effects, so far not known, on the smooth muscles, apart from the effects already described. The results from the answers to this question might make it easier to explain its multifarious clinical effects, especially when used externally. Two types of investigations were carried out. To begin with, the effects of rosemary oil through the [[alpha].sub.1.2] adrenergic receptors, [beta] adrenergic and [D.sub.2] dopamine receptors were investigated. To date there is no information on this in the relevant literature. Many clinical observations, however, can only be explained by this kind of specific, i.e. receptor-transmitted effects.

In a second step we compared the familiar spasmolytic effect of rosemary oil with that of Papaverine.

Material and methods

The impact of rosemary oil on spontaneous contractile activity (SCA) of the smooth-muscle strips (SMS) from a guinea pig stomach was examined. This model enables simultaneous registration of the spasmolytic effects of rosemary oil and comparison with Papaverine (Forster et al. 1980).

Substances used

The following agonists and antagonists (Sigma, St. Louis, MO, USA) were used in the in vitro investigations: [[alpha].sub.1] agonist used are Methoxamine HCl, [[alpha].sub.1] antagonist Prazosin HCl and then [[alpha].sub.1] and [[alpha].sub.2] antagonist Benextramine 4 HCl as well as the [[alpha].sub.2] agonist p-Iodoclonidine hydrochloride antagonist the [beta] agonist Isoprotenol the [D.sub.2] Dopamin agonist Quinpirol dihydrochloride and the selective antagonist of [[alpha].sub.2] adreno receptor Rauwolscine.

Furthermore Papaverine hydrochloride (Merck, Darmstadt, Germany) and Acetylcholine chloride (acetylcholinum ophtalmicum [dispersa.sup.R], Dispersa, Germering) and substances for the Krebs' solution (Merck, Darmstadt, Germany) as NaCl, KCl, MgCl, 6[H.sub.2]O, glucose, K[H.sub.2]P[O.sub.4], NaH[CO.sub.3] and CaCl were used.

Furtheron Rosemary oil Ph.Eur. (Ch. B. 296330, WE 06100393, Typ Tunesien, Dullberg comp. Hamburg, Germany) was used. Chromatographic profile (Fig. 1): [alpha]-Pinen 12.1% (9.0%-14%), Campher 5.0% (2.5%-6.0%), [beta]-Pinen 5% (4.0%-9.0%), [beta]-Myrcen 1.5% (1.0%-2.%), Limonen 3.9% (1.5%-4.0%), Cineol 44.7% (38.0%-55.0%), [rho]-Cymen 1.4% (0.8%-2.5%), Campher 10.1% (5.0%-15.0%), Bornylacetat 1.0% (0.1%-1.5%), [alpha]-Terpineol 2.2% (1.0%-2.6%), Borneol 3.8% (1.5%-5.0%), Verbenon < 0.1% ([less than or equal to] 0.4%).

[FIGURE 1 OMITTED]

Rosemary oil solution

To 10 [micro]l rosemary oil 2 ml ethanol (98%) were added at room temperature. The transparency of the samples was determined with 1500 lux lighting. When good solubility had been established, the concentration was gradually raised from 10 [micro]l to 100 [micro]l The resultant stock solutions of 100 [micro]l rosemary oil in 2 ml ethanol (98%) (5% solution of rosemary oil in 98% ethanol) were used for further dilution processes.

Further dilution of the stock solution of rosemary oil described above was carried out with the Krebs' solution. Thereafter the real concentrations of rosemary oil in the Krebs' solution were determined by means of direct comparisons of the absorption spectra (Table 1).
Table 1

Concentrations of rosemary oil in the Krebs' solution in the organ
bath.

N  Rosemary oil  Solute volume of       Volume of rosemary oil,
   (ethanolic    rosemary oil in 20 ml  dissolved in 20 ml Krebs'
   solution 5%)  Krebs' solution        solution, rounded ([micro]l)
   ([micro]l)       ([micro]l)

1   0.005        0.005                  =0.005

2   0.050        0.050                  =0.050

3   0.100        0.100                  =0.100

4   0.500        0.330                  [approximately equal to] 0.300

5   1.000        0.381                  [approximately equal to] 0.400

6   3.000        0.565                  [approximately equal to] 0.600

7   5.000        0.812                  [approximately equal to] 0.800

8  10.000        0.967                  [approximately equal to] 1.000

9  50.000        5.077                  [approximately equal to] 5.000


Measurement of the spontaneous contractile activity (SCA) of the smooth muscles and method of preparation

The measurements were performed by means of a standardised method according to Golenhofen (Golenhofen 1976).

Preparation of the muscle fibres: The specimen of smooth muscles used for the experiments were taken from the stomach of male guinea pigs with a weight of approx. 300 g (HsdPoc:DH 300-349 g, Harlan Winkelmann Comp., Borchen, Germany). All animal experiments have been permitted by the responsible commission of the Ruhr-Univeritat Bochum, Germany.

The strips of stomach muscle are 12-14 mm in length and 1-2 mm wide and come from the corpus and antrum region of the stomach. The strips of muscle were prepared in a circular direction, starting with the serosa, along the big curvature and as far as possible in the direction of the fibres.

The organ baths are composed of (mmol/l): [Na.sup.+]143; [K.sup.+] 5.94; [Mg.sup.2+] 1.19; [Ca.sup.2+] 2.5; [Cl.sup.-] 133; H[CO.sup.-] 16.7; P[O.sub.4.sup.2-] 11.9 and glucose 11.5, according to the Krebs' solution. During the entire experimental series the solution was carbonated, which adjusted the pH value within physiological limits between 7.2 and 7.4 (7.2+/-0.8). The Krebs' solution in the organ baths is attemperated to 35[degrees]C (+/- 0.2[degrees]C).

It was measured under isometric conditions (mN).

Statistics

In order to take the preparation-specific scattering of the SCA measured values and their changes into consideration when presenting the dose-effect curves, the appropriate excitation in some N = 10 individual experiments was always given in % of the maximum contractile activity of the smooth muscle strips under the effect of [10.sup.-5] M acetylcholine (ACh).

The experimental data are processed with the help of the Statistica 4.5 program (StatSoft, Inc. Microsoft, USA). The t-Test (student) was carried out for unrelated random samples for the purpose of comparison between two groups. For comparisons between three or more groups, variance analyses (ANOVA) were used. Statistical comparisons were carried out at the 5% significance level. The results are represented as the mean value [+ or -] standard deviation. N = 7 measurements were carried out per experiment/test.

Results

Fig. 2 shows the effect of rosemary oil in concentrations between 0.001 and 5 [micro]l in 20 ml organ bath on the SCA of the SMS of guinea pig stomach. It can be seen that at concentrations of 0.01 [micro]l (in 20 ml organ bath) excitatory effects already start to occur. The maximum excitatory effects are achieved by adding approximately 0.6 [micro]l of rosemary oil. The values are over 40% of the maximum contraction, achieved with ACH [10.sup.-5] M. If the concentration of the rosemary oil is raised again to above 1 [micro]l there is a significant suppression of these excitatory effects.

[FIGURE 2 OMITTED]

Fig. 3 demonstrates the effect of 0.5 [micro]l rosemary oil on the SCA of the SMS under normal conditions (top left) and after the [[alpha].sub.2] adrenergic receptors were blocked with [10.sup.-5] M Rauwolscine (top right). It is evident that, if the [[alpha].sub.2] adrenergic receptors are blocked, the excitatory effect of rosemary oil on the SCA of the SMS is significantly smaller. Fig. 3 (bottom left) demonstrates the effect of rosemary oil when the [[alpha].sub.1] adrenergic receptors were previously blocked with [10.sup.-5] M Prazosin. If the [[alpha].sub.1] adrenergic receptors are blocked with [10.sup.-5] M Prazosin, the excitatory effect of rosemary oil on the SCA of the SMS is also significantly less pronounced. If the [[alpha].sub.1] and [[alpha].sub.2] adrenergic receptors are blocked with [10.sup.-5] M Prazosin and [10.sup.-5] M Rauwolscine, the excitatory effects of rosemary oil will be completely cut back (Fig. 3 bottom right).

[FIGURE 3 OMITTED]

Fig. 4 shows that rosemary oil (0,5 [micro]l) used in long term effects suppresses completely the activity of the [[alpha].sub.1], and [[alpha].sub.2] adrenergic receptors.

[FIGURE 4 OMITTED]

Fig. 5 shows a comparison of the spasmolytic effects of rosemary oil and Papaverine. It is evident from the graphs that the SCA of the SMS is already suppressed to 30% if 0.8 [micro]l rosemary oil are added (in 20ml organ bath). The effect of [10.sup.-5]M ACH, however, is not achieved until 5 [micro]l rosemary oil (in 20 ml organ bath) is added. Fig. 5 (below) shows that [10.sup.-4] M Papaverine completely suppresses not only the SCA of the SMS, but also the effect of [10.sup.-5] M ACH.

[FIGURE 5 OMITTED]

To make it easier to compare the spasmolytic effects of rosemary oil and Papaverine, the concentrations of rosemary oil were calculated in Mol, analogous to the average molecular weight of the main constituents.

Discussion

These results leads one to assume that rosemary oil produces an agonistic effect on the [[alpha].sub.1] und [[alpha].sub.2] adrenergic receptors. However, the excitatory effects can only be registered in concentrations up to 0.6 [micro]l in 20 ml organ bath (or 30[micro]l/I), and the observed results are reversible. At these concentrations the spasmolytic effect of rosemary oil cannot be registered via the SCA of the SMS.

The results shown in Fig. 3 confirm our assumption concerning the effects of rosemary oil on the [[alpha].sub.1] and [[alpha].sub.2] adrenergic receptors. The results demonstrate that because of long term effects of the rosemary oil (0.5 [micro]l) on the SCA tachyphylaxis is induced and subsequently the effectivity of the receptors is decreased (Lansberg and Young 1998).

At higher doses the spasmolytic effect of rosemary oil on smooth muscles can be registered. Even at concentrations of only 1 [micro]l (in 20 ml organ bath) this spasmolytic effect brings about a 100% inhibition of the SCA of the SMS and reduces the effects of [10.sub.-5]M ACH to 26%. Doses in excess of 5 [micro]l (in 20 ml organ bath) not only totally inhibit the SCA of the SMS and the effects of [10.sub.-5]M ACH, after 10 min of exposure they cause an irreversible contracture.

The described effects of rosemary oil can bring about diverse effects in the tissue and explain two effects from the external clinical application/use of rosemary oil. For example, activation of the [[alpha].sub.2] adrenergic receptors in the neurons may cause the release of Noradrenalin to be inhibited. As a consequence vasodilatory effects arise that are comparable to low doses of p-iodoclonidine (Sadjak et al. 2006). If the release of Noradrenalin is inhibited, pain transmission is suppressed. This means that this mechanism may enable pain-relieving effects from the use of rosemary oil.

The pronounced spasmolytic effect of rosemary oil on the smooth muscles certainly causes increased blood flow in the tissue.

The next step is to examine specific effects of rosemary oil on the [[alpha].sub.1] and [[alpha].sub.2] adrenergic receptors for their clinical relevance.

Acknowledgement

We thank Truw Company, Gutersloh, for the financial support for our research work.

References

Beer, A.-M., Lukanov, J., Sagorchev, P., 2007. Effect of thymol on the spontaneous contractile activity of the smooth muscles. Phytomedicine 14, 65-69.

Cabo, J., Crespo, M.E., Jimenez, J., Zarzuelo, A., 1986. The spasmolytic activity of various aromatic plants from the province of Granada. The activity of the major components of their essential oils. Plant. Med. Phythother. 10 (3), 213-218.

Forster, H.B., Niklas, H., Lurz, S., 1980. Antispasmodic effects of some medicinal plants. Planta Med. 40, 309-319.

Golenhofen, K., 1976. Spontaneous activity and functional classification of mammalian smooth muscle. In: Bulbring, E., Schuba, M.F. (Eds.), Physiology of Smooth Muscle. Raven Press, New York, pp. 91-97.

Imaseki, I., Kitabatake, Y., 1962. Studies on effect of essential oils and their components on the isolated intestines on mice. J. Pharm. Soc. Jpn. 82 (9), 1326-1328.

Lansberg, L., Young, J.B., 1998. Physiology and pharmacology of the autonomic nervous system. In: Harrison's Principles of Internal Medicine, 14th ed., McGraw-Hill.

Santoyo, S., Cavero, S., Jaime, L., Ibanes, E., Senorans, F.J., Reglero, G., 2005. Chemical composition and antimicrobial activity of Rosmarinus officinalis L. essential oil via supercritical fluid extraction. J. Food Prot. 68 (4), 790-795.

Sadjak, A., Wintersteiger, R., Zakel, D., Wenzl, T., Lankmayr, E.P., Gferer, E., Likar, R., 2006. Peripheral analgesic effect of intra-articularly applied clonidine. Der Schmerz 20 (4), 293-299.

Schilcher, H., Kammerer, S., Wegener, T., 2007. Leitfaden Phytotherapie. 3. Auflage. Elsevier, Urban & Fischer, Munchen, pp. 201-202.

Taddei, I., Giachetti, D., Taddei, E., Mantovani, P., Bianchi, E., 1988. Spasmolytic activity of peppermint, sage and rosemary essences and their major constituents. Fitoterapia 59, 463-468.

Wagner, H., Wierer. M., Bauer, R., 1986. In vitro-Hemmung der Prostaglandin-Biosynthese durch atherische Ole und phenolische Verbindungen. Planta Med. 52, 184-187.

P. Sagorchev (a), J. Lukanov (a), A.M. Beer (b), *

(a) Department of Biophysics, Med. University Plovdiv, Bulgaria

(b) Department of True naturopathy, Blankenstein Hospital, Im Vogelsang 5-11, 45527 Hattingen, Germany

* Corresponding author. Tel.: +49 2324 396487; fax: +49 2324 396497.

E-mail address: andre.beer@klinik-blankenstein.de (A.M. Beer).

0944-7113/$ - see front matter [C] 2009 Published by Elsevier GmbH.

doi: 10.1016/j.phymed.2009.09.012
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Title Annotation:Short Communication
Author:Sagorchev, P.; Lukanov, J.; Beer, A.M.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:4EUGE
Date:Jul 1, 2010
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