Relaxant Effect of the Ethanol Extract of Helichrysum plicatum (Asteraceae) on Isolated Rat Ileum Contractions

Helichrysum plicatum (Turkish Helichrysum) has been used in folk medicine for the treatment of gastric and hepatic disorders. The aim of the present study was to examine the relaxant activity of an extract of H. plicatum flowers on isolated rat ileum. Segments of ileum of rats were suspended in an organ bath. Cumulative concentrations of H. plicatum ethanol extract induced a relaxant effect on spontaneous rat ileum contractions. H. plicatum extract caused a mean contractile response of 81.68 ± 6.17% (at a dose of 0.01 mg/mL) and 30.08 ± 9.07% (at a dose of 1 mg/mL). A similar effect was observed with papaverine (0.01–3 μg/mL). H. plicatum extract (0.01–1 mg/mL) relaxed high K+ (80 mM) precontractions, an effect similar to that caused by papaverine (0.01–3 μg/mL). The plant extract (0.03–0.3 mg/mL) also induced a significant depression of the cumulative concentration response curve for acetylcholine (5–1500 nM) (p < 0.01). Atropine (140 nM) abolished the acetylcholine effect. The extract (0.03–0.3 mg/mL) reduced the histamine (1–300 nM) and BaCl2 (3–900 μM) induced contractions (p < 0.01). Our results showed the relaxant effect of the ethanol extract of Helichrysum plicatum flowers on the isolated rat intestine Extract of H. plicatum can inhibit the spontaneous ileum contractions and contractions induced by acetylcholine, histamine, barium and potassium ions.


Introduction
Plants are sources of many medicinal drugs. For centuries, herbs have been used in traditional medicine to treat of many gastrointestinal disorders. In recent times numerous scientific studies have been performed to test the potential effect of plant extracts on intestinal contractions [1]. However, the mechanism of action by which these plants exert their therapeutic effects has not been completely elucidated [2].
The genus Helichrysum (Asteraceae) comprises approximately 500-600 species. Species from the genus Helichrysum are used in Europe and Africa in the treatment of various medical conditions [3]. Helichrysum spp. have been used in folk medicine for at least 2,000 years as choleretics, cholagogues, hepatoprotectives and for stimulation of the secretion of gastric juice [4,5]. Some members of the genus are known for their anti-inflammatory, anti-allergic and antibacterial properties [6]. In Turkish folk medicine H. plicatum has been used as a diuretic, lithagogue, and for stomach ache [7]. H. plicatum has been used in Serbian and Macedonian folk medicine for the treatment of gastric and hepatic disorders. Moreover, the antidiabetic, and antioxidant activity of this species have also been reported [8].
The aim of the present study was to evaluate the possible spasmolytic activity of the ethanol extract of Helicrisum plicatum on rat ileum, since there is no data of the physiological effects of the extract on isolated rat intestine.

Results and Discussion
The results presented in Figure 1 demonstrate that H. plicatum extract in cumulative concentrations (0.01-1 mg/mL) induced a concentration-dependent inhibition of the spontaneous contractions of the rat ileum. Extract of H. plicatum caused a mean contractile response of 81.68 ± 6.17% (at a concentration of 0.01 mg/mL) and 30.08 ± 9.07%, at a concentration of 1 mg/mL (p < 0.01). The calculated EC 50 value was 0.13 ± 0.01 mg/mL. A similar effect was observed with papaverine (EC 50 value was 0.06 ± 0.003 μg/mL, Figure 2).
The extract of H. plicatum (0.01-1 mg/mL) relaxed the tonic contraction induced by KCl in a concentration dependent manner, with an EC 50 value of 0.22 ± 0.03 mg/mL ( Figure 1). Papaverine (EC 50 0.09 ± 0.008 μg/mL) was also used as a positive control. Acetylcholine caused a concentration dependent contraction of rat ileum. Plant extract (0.03-0.3 mg/mL) induced a significant depression of the cumulative concentration response curve for acetylcholine (p < 0.01). Extract of H. plicatum caused a modification of the EC 50 of acetylcholine from 58.86 ± 4.58 nM (in the absence of the extract) to 2,624.24 ± 104.69 nM, in the presence of the extract at a concentration of 0.3 mg/mL (Table 1).   Pretreatment of the tissue with atropine (140 nM) abolished the acetylcholine effect ( Figure 3).   The extract of H. plicatum was studied for its possible spasmolytic effect on rat intestine. Isolated ileum suspended in an organ bath showed spontaneous contractile activity. Our in vitro studies indicate that the plant extract produces relaxant effects on rat ileum spontaneous contractions. The effect was concentration-dependent and reversible after washing the tissue with Tyrode`s solution. The observed spasmolytic activity of H. plicatum extract was less potent than that of papaverine, a nonspecific smooth muscle relaxant. Papaverine is a dual inhibitor of Ca 2+ influx [9] and phosphodiesterase inhibitor [10][11][12][13].
The enteric nervous system is considered to be an independent nervous system that controls and coordinates gastrointestinal motility. Gastrointestinal motility is regulated by numerous mediators, mainly acetylcholine, histamine, 5-hydroxytryptamine, bradykinins, prostaglandins, substance P and cholecistokinins which achieve their contractile effects through an increase in cytosolic Ca 2+ [14,15]. To clarify the possible underlying mechanism, we investigated the influence of the extract on acetylcholine, histamine, BaCl 2 and KCl-induced smooth muscle contraction.
It was found that the extract of H. plicatum exhibited a significant depressive effect on the cumulative concentration response curves for acetylcholine in the isolated rat ileum. Pretreatment with atropine abolished the contractile effect of acetylcholine. Acetylcholine, a neurotransmitter, is released by the parasympathetic nervous system, and plays an important physiological role in the regulation of gut movements [16]. The acetylcholine induced contractions of the rat ileum involve two different mechanisms coupled to muscarinic receptors. One mechanism activates non-selective cation channels in the plasma membrane, which results in membrane depolarization. The depolarization stimulates Ca 2+ influx through voltage-gated Ca 2+ channels. The other mechanism activates contraction by the release of intracellular calcium [17].
One of the possible mechanisms for the spasmolytic activity of the extract could be mediated through the inhibition of histaminic receptors. In our study the extract of H. plicatum inhibited the contractions of rat ileum exerted by histamine in a concentration dependent manner. Histamine is known to cause contraction of smooth muscle of the gastrointestinal tract. Application of histamine to intact smooth muscle produced a concentration-dependent membrane depolarization and increased excitability [18,19].
To specify the spasmolytic activity of H. plicatum extract, we tested its effect on the contraction of isolated rat ileum induced by BaCl 2 and KCl, to find out if H. plicatum is a musculotropic substance. Barium ion (Ba 2+ ) has been known to induce contractions in smooth muscle tissues. Ba 2+ depolarizes the smooth muscle membrane and opens the voltage-dependent Ca 2+ channels resulting in a Ca 2+ influx [20]. H. plicatum extract produced a statistically significantly inhibition of the the contractions induced by BaCl 2 .
According to the literature a lot of plant secondary metabolites can mediate spasmolytic effects through inhibition of the influx of calcium into the cell. High K + stimulation, which provokes membrane depolarization, is the most common method for the introduction of Ca 2+ into cell without receptor stimulation [21]. High concentration of K + cause smooth muscle contractions through opening voltage-dependent Ca 2+ channels, thus allowing an influx of extracellular Ca 2+ causing a contractile effect [22]. A substance causing inhibition of high K + -induced contractions is considered as a blocker of calcium influx [23].
Karaki et al. [20] have researched contractions of intestinal smooth muscle induced by Ba 2+ and high concentrations of K + . They concluded that both contractions are due to influx of Ca 2+ . The extract of H. plicatum exhibited an inhibitory effect on intestine contractions induced by BaCl 2 and high concentrations of K + . According to Gilani et al. [24] different plant extracts usually mediate their spasmolytic action through blockage of Ca 2+ influx. This indicates that the plant extract is preventing the increase of calcium ions and muscle tension [25][26][27]. The extract of H. plicatum caused relaxation of the K + -induced contractions, suggesting that the spasmolytic effect is possibly mediated through calcium channel blockage.
There is growing evidence that the spasmolytic effect of the extract is associated with the presence of phenolic compounds. Flavonoids are one of the most numerous and widespread group of phenolics in higher plants. Some of them inhibit intestinal motility in vitro. Quercetin produces relaxation in ileum contracted by KCl [28]. Apigenin and luteolin inhibited the contractions of isolated intestinum [29,30]. These substances have been reported to exhibit calcium antagonist and anticholinergic activities [31,32].
The medicinal properties of the genus Helichrysum are mainly attributed to the presence of flavanoids [33,34]. Our previous study showed that ethanol extracts of H. plicatum has high flavonoid heteroside content. Naringenin glycosides, apigenin-7-glucoside, quercetin-and kaempferol-3glucoside were identified in notable concentration. Also, the amount of total phenolics varied from 101.1-266.9 mg GAE/g DW in different ethanolic extracts [35]. Based on this report, the spasmolytic activity of the ethanol extract of H. plicatum studied here could be attributed to flavanoids and other phenolic compounds. The key role of phenolic compounds as spasmolytic agents is emphasized in several reports. Our results are also in agreement with Mulatu [36] and Palacios-Espinosa [37] who have reported the spasmolytic activity of other plants species belonging to the same (Asteraceae) family.

Plant material
Flowers of H. plicatum were purchased from a commercial supplier (Agroherbal, Albania, batch number 29470605), in July 2007. Pulverization (sieve numbers according to European Pharmacopoeia 6.0. 2008) was carried out just before analysis. Flower heads of H. plicatum were extracted with aqueous ethanol (1:1) by triple percolation. Re-extraction were done by ethyl acetate-ethanol and then the extract was evaporated under vacuum at 60 °C to obtain a yellow-orange dried powder. The obtained extract was kept at 4 o C. For experimental purposes the plant extract was first dissolved in ethanol (20% m/m), than diluted with distilled water to the appropriate concentration. Ethanol, at the same concentrations, had no effect on intestine contractility in the control experiments.

Animals
The research was conducted in accordance with the European Council Directive of November 24, 1986 (86/609/EEC). Male and female Wistar albino rats (200-250 g), 3-4 months of age, were used after a 24 h fasting with free access to water. Ileums were prepared and placed in 10 mL tissue baths containing Tyrode`s solution at 37 ºC and constantly aerated with a carbogen [38]. The preparations were equilibrated for 30 min. In the experiments 30 rats were sacrificed. The change of intestinal contractility was recorded using a transducer (TSZ-04-E, Experimetria Ltd, Budapest, Hungary), recorded and analyzed with a SPEL Advanced ISOSYS Data Acquisition System (Experimetria Ltd, Budapest, Hungary).

Experimental protocol
The tissues had been pretreated with the ethanol extract of H. plicatum (0.01-1 mg/mL). Papaverine (0.01-3 μg/mL) was used as control. In the second series of experiments, isolated intestinal segments were contracted by depolarization with 80 mM KCl. After tonic contractions were obtained, extract of H. plicatum (0.01-1 mg/mL) was cumulatively added at 15 min intervals. Papaverine (0.01-3 μg/mL) was added to the bath. Increasing concentrations of acetylcholine (5-1500 nM), histamine (1-300 nM) and barium chloride (3-900 μM) were added to the organ bath cumulatively to generate full concentration response curves. Then concentration response curves were obtained in the presence of the extract of H. plicatum (0.03-0.3 mg/mL) in the organ bath.

Statistical analysis
Mean and standard error values were calculated for each group of results (n = 6 for each set of experiments) and significance of differences between the means were determined by the Student`s ttest. A probability value of p < 0.05 or less was noted as indicative of significance. An EC 50 value (concentration of drugs causing half-maximal responses) was established by regression analysis.

Conclusions
The results obtained from the present study indicate that the ethanol extract of Helichrysum plicatum flowers showed a relaxant effect on isolated rat intestine. The extract of H. plicatum can inhibit spontaneous ileum contractions and contractions induced by acetylcholine, histamine, barium and potassium ions. The therapeutic effectiveness in the treatment of gastrointestinal disorders and use in traditional medicine of this plant could be due to its spasmolytic effect.