Spasmolytic Effect of Petroselinum crispum (Parsley) on Rat’s Ileum at Different Calcium Chloride Concentrations

MATERIALS AND METHODS

Plant extract: This protocol has been done in April, 2006 until May, 2007. Seeds of parsley were prepared by Jahad Research Center (Ahwaz) and identified by Department of Biology, Shahid Chamran University, Ahwaz, Iran. The samples were ground to powder, 1 g of powder was extracted using 10 mL of ethanol-distilled water (8:2 w/v), centrifuging for 15 min and the collecting the supernatants. This process was repeated three times. The ethanol was then removed by evaporation (Patumi et al., 1990; Seyyednejad et al., 2001).

Drugs and solution: The following drugs were used for the experiments: KCl (Merck), CaCl₂ (Merck), parsley (alcoholic extract). Chloride potassium (KCl) was made up as 60 mM (Nasu et al., 1994) stock solution in Tyrode. The plant extracts were made up 0.1 g/1 cc Tyrode as stock solution. Various concentration of CaCl₂ were made up as (0.225, 0.45, 0.9, 1.8 and 3.6 mM) stock, dilution in Tyrode (without CaCl₂ and high concentration of K⁺). Tyrode’s solution composed of (Mm): NaCl (139.9), KCl (2.68), CaCl₂ (1.8), MgCl₂ (1.05), NaHCO₃ (11.9), NaH₂PO₄ (0.42) and glucose (5.55), was made up in distilled water (Sadraie et al., 2001, 2003). All chemical were purchased from Merck.

Experimental procedure: Male Wistar rats (225±25 g) were killed by cervical dislocation and a portion of ileum was isolated, removed and suspended in a 10 mL organ chamber containing Tyrode solution at 37°C. For the experiments done in Ca free solution we used Tyrodes solution without CaCl₂ and high concentration of potassium (60 mM). The connected tissue was carefully trimmed from the tissue and placed in Tyrode solution at 37°C and bubbled with air. Muscle preparations were set at a resting tension of 1 g and allowed to equilibrate for 1 h in tyrode solution. Isotonic contractions, elicited by KCl and CaCl₂ were recorded using a Harvard isotonic transducer and displayed on a Harvard Universal Oscillograph pen recorded device. Drugs were added directly to the organ bath in volumes usually not exceeding 5% of bath volume (10 mL organ bath). A contraction-response curve was also obtained by cumulative addition of drugs at 5 min intervals after addition of 60 mM KCl. Each drug concentration was at 5 min in contact with the tissue before their effects were evaluated.

Measurements and statistical analysis: Contractions were measured as maximum changes in tension from pre-drug baseline within the contact time or as the area under curve produced by tissue contraction at 5 min intervals just before addition of the next concentration of the test drug and expressed as percentage of control or maximum induced response for each tissue. Mean and standard error of mean (SEM) values were calculated for each group of results and significance between the means was calculated by two-tailed paired student’s t-test or one-way analysis of variance (ANOVA). Values were considered to be significantly different from control when p<0.05.

RESULTS

The effect of extract on ileum’s contraction-induced by KCl: Rats ileum suspended in Tyrodes solution shows irregular spontaneous contractile activity, which attenuates by changing of bath fluid. KCl 60 Mm (non-receptor stimulation for opening Ca channels) produced a sustained tonic contraction, which was maintained during the course of experiments. After administration of 60 mM KCl to the bath solution, various cumulative concentrations of extract (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 and 0.8 mg mL‾¹) were added to the bath. The extract to the dose-dependent manner significantly (n = 7, p<0.001) inhibited the ileum contraction induced by 60 mM KCl (Fig. 1). These inhibitory effects of the parsley extract could be seen within 5 min of contact with the tissue and were maintained as long as it was present in the bath.

Fig. 1:	The effect of cumulative concentration of alcoholic Parsley seed extract on KCl-induced contraction in the adult male rats ileum (n = 7). ***: p<0.001. A: Histogram, B: One sample of oscillogram

Fig. 2:	The effect of 0.8 mg mL‾1 extract on KCl-induced contraction in the adult male rat’s ileum

As is shown in Fig. 2, administration of 0.8 mg mL‾¹ extract, to become apparented maximum response relaxation in ileum but reduced density effect of it with pass a time. On the other hand, in another experiment KCl were added to the bath in present of each concentration of extract and them washing it for 15 min (non-cumulative).

Fig. 3:	The effect of non-cumulative concentration of alcoholic Parsley seed extract on KCl-induced contraction in the adult male rat’s ileum (n = 7). *: p<0.05, ***: p<0.001

These results showed that, utilization from KCl could contractions on ileum for several time which were no significant difference between them (n = 7, p<0.001) (Fig. 3).

The effect of extract on ileum before effect of KCl: In this stage, ileums tissue incubation with various concentration of extract (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 and 0.8 mg mL‾¹), then KCl 60 mM were added to the bath.

Fig. 4:	The effect of various concentration of alcoholic parsley seed on adult male rat’s ileum, before influence of KCl 60 mM (n = 7). **: p<0.01, ***: p<0.001; (A) Histogram and (B) One sample of oscillogram

Fig. 5:	The effect of various concentration of CaCl2 on contraction of adult male rat’s ileum at absent of extract (n = 7). *: p<0.05, ***: p<0.001; (A) Histogram and (B) One sample of oscillogram

Fig. 6:	The effect of various concentration of CaCl2 on contraction of adult male rat’s ileum at present of extract (n = 7); ***: p<0.001; (A) Histogram and (B) One sample of oscillogram

The results indicated that the presence of alcoholic parsley seed extract, prevented from contraction-induced by KCl (n = 7, p<0.001) (Fig. 4).

The effect of various concentration of calcium on ileum’s contraction in absent and present of extract: To investigate the possible mechanism responsible for the relaxation response induced by extract in the ileum, cumulative addition of various concentrations of CaCl₂ to the bath tissue that including high potassium and without CaCl₂, caused strong contraction (n = 7, p<0.001) (Fig. 5).The following this stage before adding CaCl₂, application different concentration of extract (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7 and 0.8 mg mL‾¹) at intervals of 5 min. The present results have provided evidence indicating that although in the low dose of extract, CaCl₂ caused contraction on ileum, but this effect were lesser than contractile response tissue in absent extract (n = 7, p<0.001) (Fig. 6).

DISCUSSION

This experiment showed that, alcoholic extract of Petroselinum crispum in a concentration-dependent manner inhibit rat ileum contraction induced by 60 mM KCl (Fig. 1). In addition, The inhibitory effects of all extracts were reversible after washing the tissue with fresh Tyrodes solution. These results confirm that the inhibitory effects observed, was not arising since fatigue smooth muscle ileum (Fig. 3). The parsley essential oil in current analysis characterized by the dominant presence of five Substance: myristicin, apiol, α-pinene, β-pinene and 1- ally 1, 2, 3, 4, 5-tetramethoxy-benzene (Hui et al., 2006). Essential oil of Alpinia speciosa schum (EOAS) possesses both relaxant and antispasmodic actions in ileum (Bezerra et al., 2000). Some reports indicated that the effects of the Plectrantus barbatus essential oil (PBEO) decreased the basal tonus of the ileum with a maximal response compared with inhibition achieved with α-pinene. They show that PBEO has intestinal relaxant and antispasmodic activity and suggest that it seems to occur down stream of calcium entry or release from internal stores. The main active principle for its relaxant and spasmolytic activity seems to be α-pinene (Camara et al., 2003). α-pinene and β-pinene two components of the Ferula gummosa essential oil (EGEO) and the hydroalcoholic extract had clear inhibitory effect on tonic contraction induced by KCl (Sadraie et al., 2001). The inhibitory effect of Teucrium polium essential oil on smooth muscle is due to an effect on calcium channels (Aqel and Ghavaibeh, 1990). The inhibitory effects of essential oil from various plants with different components are qualitatively similar, they may all have a similar mechanism of action (Sadraie et al., 2001). The aqueous extract of Pavetta crassipes leaves exerted, a concentration-dependent inhibition of the spontaneous motility or elevated tone in these preparations. Their results indicate the presence of biologically active substance whose action might be mediated through calcium channels. A preliminary phytochemical screening of the leaf extract of P. crassipes revealed the presence of flavonoids, tannins an anthraquinones as possible candidates for such inhibition substances (Amos and Okwuasaba, 1998). The inhibitory effect of quercetin on the guinea-pig ileum contractile response, at least partially, due to blockade of calcium concentration by KCl. Quercetin inhibits the contractile response of ileum, decreasing the calcium concentration available for contractile machine (Galvez et al., 1996). There was a report that hydroalcoholic extract of Pycnocycla spinosa was shown to have spasmolytic action in vitro and antidiarroheal effect in vivo. The hydroalcoholic extract of P. spinosa is composed of alkaloid, flavonoid and saponin-rich components. Antispasmodic action of hydroalcolic extract of P. spinosa could be due to components in the alkaloid-rich fraction and to a lesser extract, to its flavonoid-rich fraction (Sadraie et al., 2003). Many scientists in their investigations have been shown that vasorelaxant action of the aqueous leaf extract of Persea americana on isolated rat aorta. The extract reduced vasoconstriction probably by inhibiting calcium influx through calcium channels (Owolabi et al., 2005). However, it seems that relaxation effect observed on rat isolated ileum contractions, was induced by present components (essential oil, α-pinene, β-pinene) of hydroalcoholic extract of Petroselinum crispum.

In order to survey mechanism the action of extract, the role of calcium on contraction induced by KCl was investigated. Also our results showed that various concentration of Petroselinum extract decreased the KCl-induced contraction in Tyrode solution, which including high K⁺ (60 mM) and without CaCl₂ (Fig. 5). This result is similar as the result of many other investigators. For instance, as shown by Gharib Naseri et al. (2006), spasmogenic effect of cumulative concentration of calcium on rat colon in Ca-free with rich potassium Tyrode solution, was reduced by the hydroalcoholic extract of the grape leaf.

The contractile activity of smooth muscle can be controlled by numerous factors, including hormones, autonomic nerves, pacemaker activity and variety of drug. Like skeletal muscle or cardiac muscle, the contraction of smooth muscle is calcium-dependent and the agents listed above cause smooth muscle contraction by increasing intracellular calcium concentration. Smooth muscle also has an intracellular membrane network of Sarcoplasmic Reticulum (SR) that serves as an intracellular reservoir for calcium. Calcium can be released from the SR into the myoplasma when stimulatory neurotransmitters, hormones or drugs bind to receptors on the sarcolemma (Watras, 2004). Ca is a fundamental second messenger in smooth muscle cells. An increase in cytoplasmic Ca concentration and binding to calmodulin and the activation of myosin light chain kinase is the primary stimulus for contraction. The Ca used in the activation of the contractile apparatus enters the cytoplasmic compartment during periods of membrane depolarization, mechanical distortion, or stimulation by agonists. Contraction of smooth muscle is regulated by cytosolic Ca levels and sensitivity to Ca of the contractile elements in response to changes in the cell (Grasa et al., 2004; Ratz et al., 2005). There is evidence that there can be significant variation in the degree of participation of extracellular and intracellular Ca in smooth muscle contraction. Other results indicate that extracellular Ca participates in spontaneous activity and enters cytosol by L-type voltage-dependent Ca channels (Grasa et al., 2004). Calmodulin (CaM) plays important roles for contractile activity in smooth muscles. In gastrointestinal smooth muscles (GISM), CaM functions as the regular of contractile behavior and electrical rhythmicity. In GISM, contraction is mediated by the initial [Ca]i transient, resulting in Ca/Calmodulin-dependent activation of myosin light chain (MLC) kinase, phosphorylation of MLC20 and interaction of actin and myosin, whereas relaxation is mediated by cAMP-and or cGMP-dependent protein kinase which inhibit the initial [Ca]i transient and reduced the sensitivity of MLC kinase to [ca]i (Ohya and Horowitz, 2002). Studies done in 1984, with the use of intact smooth muscle tissue, showed that GPCR agonist (G protein-coupled receptors) can produce greater increase in force for a given increase in [ca]i than KCl. KCl-induced contraction has long been known to be due to membrane depolarization causing Ca entry through voltage-operated Ca channels (VOCCs), activation of Ca-dependent MLC kinase and increase in MLC phosphorylation (Ratz et al., 2005). Also there is evidence for that in guinea pig ileum and Taenia coli, high K⁺ elicits an increase in [Ca]i and transient contractions. The spontaneous movements in longitudinal muscle of sheep duodenum persist in Ca-free solutions with EDTA for 20 min. Verapamil, nifedipine and diltiazem all of which are Ca antagonists, reduce the mechanical activity of rabbit ileum, rat duodenum, sheep duodenum and canine ileum. Results demonstrated that in the spontaneous contractions of rabbit intestinal muscle extra cellular Ca plays principles role in longitudinal and circular muscle, whereas both extra cellular and intra cellular Ca participate in the Ach and KCl induced contractions in rabbit small intestine (Grasa et al., 2004). However, in order to suggest which the inhibitory effect on the contraction of ileum produced by the alcoholic extract of the parsley seeds results from blocking voltage-gated calcium channels. Although the relaxation role of parsley seed has been demonstrated, still the chemical nature and identity of the active ingredient(s) are unknown and will be investigated in a follow-up project.