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The Role of Histaminergic H2 Receptors on Spasmolytic Activity of Hydroalcoholic Extract of Parsley (Petroselinum crispum) Seeds in Isolated Rat’s Ileum

Objective: The long traditional history of anti-spasmodic effects of Petroselinum Crispum on the gastrointestinal system, and considering histamine receptors in rats ileum, to elucidate the role of the histamine receptor in function relate intestinal motility in this study investigate the effects of histamine and antagonists H2 receptor (Cimetidine) on contractions of adult male rat’s ileum. Methods: In this study, 2 cm of terminal isolated adult male rat’s ileum in an organ bath containing the oxygenated Tyrode solution (370c, PH = 7.4) was used and placed under 1 gram tension contraction induced by 60mM potassium chloride. The effect of parsley extract was measured in presence of histamine and antagonists H2 receptor (cimetidine) and their effects on the rat،s ileum contractions were recorded by using Harvard oscillograph. Results: The parsley extract reduced ileum contraction induced by KCl in a dose-dependent manner (P<0.001, n = 7). Spasmolytic effect of extract after tissue exposure to histamine (0.1 mg/ml) as an agonist and cimetidine (0.004 mg/ml) did not decrease ileum contractions. Conclusion: Although Parsley seeds extract reduced rat ileum contraction, it seems that H2 histamine receptors (cimetidine) are not involved in this activity.

Plants are sources of many medicinal drugs. For centuries, herbs have been used in traditional medicine to treat of plenty gastrointestinal disorders 3 . Some herbal teas act as antispasmodics 4 .
Gastrointestinal disorders are the most common problems affecting the general population. Synthetic drugs are present, however, with low efficacy and high side effects 5 . Disorders such as diarrhoea are a major problem in the developing countries. Therefore it was the cause of millions of deaths 2 .
One of the plants that are traditionally used in the treatment of gastrointestinal disorders is Petroselinum crispum, Parsley is a culinary and medicinal herb of the Apiaceae family that grows up to 30-110 cm high. In folk medicine, different parts of it have been used for different purposes the aerial part of P. crispum is used to treat hemorrhoids, the stem for urethral inflammation, and the root is used to pass kidney stones and improve brain function and memory. It also plays the role of carminative stomachic, abortifacient, and nutritive agent. Numerous Studies have shown that P. crispum has hypoglycemic, diuretic, hypolipidemic, antimicrobial, and anticoagulant and hepatoprotective properties 6 .
In the previous study, we reported that the inhibitory effect of parsley seed extract on ileum contraction was likely due to the blocking of voltage-gated of calcium channels 8 . Gastrointestinal motility is an integrated process which includes a myoelectric contractile activity, tone, compliance, and transit. Those aspects of motility can be generated and modulated by local and circulating neurohumoral substance 9,10 .
Motor activity of smooth muscles is affected by nerve agents, chemical mediators, drugs and muscle tension. These factors through affecting the cellular mechanisms of muscle activity cause muscle movements. The factors affecting the muscle ion channels, calcium homeostasis, and cytosolic enzymes change the motor activity of the muscles 11 .
The classical signaling pathway through a contraction is evoked by the entrance of ca +2 smooth muscle, ca2+ calmodulin (Cam) complex formation and, finally, Myosin Light Chain (MLC) phosphorylation to induce smooth muscle contraction 12 .
The membrane of smooth muscles has calcium channels dependent on higher voltage in comparison with skeletal muscles that have sodium channels dependent on much lower voltage; therefore, mainly the current of calcium ions through sodium and calcium slow channels into the fiber is responsible for the production of the contraction activity 11 . 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 cholecystokinins which achieve their contractile effects through an increase in cytosolic Ca2 3 .
Histamine is one of the aminergic neurotransmitters and plays an important role in the regulation of several (photo) physiological processes 13 .
Distinct effects are dependent on receptor subtypes and their differential expression. Within the gastrointestinal tract, histamine is present at relatively high concentrations particularly during inflammatory responses 14 . Histamine is released by degranulation in response to different stimulants, such as anaphylatoxins or cross-linking of their high affinity IgE receptors, and has multiple physiological effects such as increasing vascular permeability, causing smooth muscle contraction, and activation of nerves in immediate hypersensitivity reactions. Many of physiological effects of histamine are mediated through four histamine receptors, H1-H4, expressed on multiple cell types 15 . That belongs to the superfamily of G-protein-coupled-receptor (GPCR) 16 .
Previous observations indicate that histamineinduced contraction of intestinal smooth muscle via stimulation of H1-receptors. H1 receptor induced by histamine provokes excitatory responses, while stimulation of the H2 receptor provokes inhibitory actions on various smooth muscle tissues 17 . The H1 receptor is associated with the phospholipase c-catalysed formation of inositol 1,4,5 triphosphate (IP3) and 1,2 Diacylglycerol (DAG). Histamine induces production of inositol phosphates in several tissues (including the brain, airway, intestinal and vascular smooth muscle) via G aq protein activation. In other tissues, activation of H1 receptors can also stimulate adenylyl cyclase and formation of cAMP in any case; alternative signaling pathways can be mediated by the histamine H1 receptor. The recent result indicated that the function of H2 receptors (H 2 R) is mediating the increase of intercellular ca2 concentration and/or IP3 levels were observed. Cimetidine (H2 antagonist) was able to block that effect of histamine on the stomach and the heart 13 and inhibited the contractile response of the ileum to dimaprit 18 . H2R is coupled both to adenylate cyclase and to phosphoinositide second messenger systems via separate GTP-dependent mechanisms. Receptor binding stimulates activation of Protein Kinase C (PKC) and p70S6 kinase. Thus, H2Rmediated impacts on cAMP accumulation have been well documented and had been demonstrated in brain slices, gastric mucosa, fat cells, cardiac myocytes, vascular smooth muscle, basophils and neutrophils 16 .
Despite a considerable number of reported studies for parsley extract, the mechanism of its effect on ileum smooth muscle has not yet been investigated. So the present work was undertaken to evaluate the role of H2 receptors on the spasmolytic effect of parsley seeds extract in rat ileum and to evaluate its clinical benefits in gastrointestinal disorder.

Plant Extract
P. crispum Latifilum seeds were collected in (Octobr 2015) form of the Behbahan and identified by Department of Biology Shahid Chamran University, Ahwaz, Iran. To prepare the hydroalcoholiclic extract of P. crispum the sample was washed and air-dried under shade, graded by an electric blender and powder (20 g) was extracted by the macerated method using 70% alcohol (100 ml) for 72 h at room temperature and were shaken several times every day. The mixture was then filtered (Whatman No.1) and the solvent evaporated (yield: 7.72 g). The extract powder was stored at 4º C until being used 8,19 .

Animal and Ileum Preparation
Male Adult Wistar rats (225±25) g used and kept at 20-24ºC and 12h/12h light/dark cycle and free access to food and water. The rats were housed in cages with the mesh bottom to prevent coprophagy and deprived of food for 24 h before the experiment but had free access to water. Animals were killed by cervical dislocation, and after the laparotomy, strip (2 cm) of ileum was dissected from 2 cm above the ileocaecal junction and intraluminal content flushed out with the cooled oxygenated Tyrode solution. The tissue was mounted in an organ bath (10 ml) containing Tyrode solution (37ºC, pH 7.4) between two stainless steel hooks and subjected to continuous air bubbling. The lower hook was fixed at the bottom of the organ bath and the upper one was connected to an isotonic transducer (Harvard Transducer). The ileum contractions were recorded, under 1 g resting tension, following 60 min for equilibrium period. During this period, the organ bath solution was refreshed every 15 min. Isotonic contraction, elicited by Kcl were recorded using a Harward isotonic transducer and displayed on a (Harward universal Oscillograph UK) pen recorded device. A contraction -response curve was also obtained by cumulative addition of 60mM Kcl. Each extract concentration had 5 min of contact with the tissue before effects were evaluated.

Effect of Extract on KCl-induced Ileum Contraction
Rat ilea suspended in tyrode's solution showed irregular spontaneous contraction activity, which was attenuated by changing the bath fluid. 60mM KCL non-receptor stimulation for opening ca2+ channel produced a sustained tonic contraction, which was maintained during the course of the experiment. After administering of 60 mM KCL to the bath solution, various concentration of extract (0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 mg/ml) was added sequentially to the bath.

Effects of Histamine (agonist)
In the other stage of the experiment, to investigate the role of histamine in the presence of parsley seeds extract, after recording the contractionary effect resulted from histamine on the isolated tissue then without washing, parsley seeds extract ( 0.2mg/ml ) were added to the organ bath.

Effect of an H2-Receptor Antagonist
In separate experiments to investigate the possible mechanisms of the relaxation response induced by parsley seeds extract, the strip of ileum was equilibrated and contracted with 60 mM KCl, then after 30 min tissue incubations with Cimetidine (0.004mg/ml) parsley seeds extract (0.2 mg/ml) were added to the bath.

Measurements and Statistical Analysis
The obtained data were analyzed by using Statistical Package for the Social Sciences software (SPSS Inc., Chicago). Changes in contractile force induced by extracts and antagonists were calculated in the form of the mean ± standard error of the mean. The statistical test of ANOVA and least significant difference post-hoc tests were used to compare the different concentrations of the extract and Student t-test was employed to compare the two groups with each other. P <0.05 was considered to be a significant difference.

Spasmolytic Effect of Extract in the Presence of Histamine
To investigate the role of histamine relaxant activity parsley seed extract after recording the effects of histamine contraction (0.1 mg/ml) without washing, concentration (0.2 mg/ml) of parsley seed extract was added to organ bath. The results showed that extracts at present histamine did not  have a significant effect on tissue contraction (n = 7) (Figure 2).

Spasmolytic Effect of Extract in the Presence of Cimetidine
Incubation of ileum preparation (30 min) with Cimetidine (0.004mg/ ml, n = 7) as a histaminergic H2 receptors antagonist, then without washing tissue stimulation by Kcl (60 mM) and then parsley seeds extract (0.2 mg/ml) was added to the organ bath. The result showed that Cimetidine did not alter the Extract (0.2 mg/ml) spasmolytic effect on ileal contractions induced by KCl (60 mM) ( Figure 3).

Discussion
The purpose of this work is to study the influence of parsley seed extract on contraction activity of rat is ileum induced by spasmogen and compare the effect of H2 receptor antagonist and histamine (general agonist). Ultimately, the goal is to investigate the benefits of this drug for gastrointestinal disorders.
Diarrhea has resulted from an imbalance between the absorptive and secretory mechanisms in the intestinal tract, accompanied by intestinal hurry, and an excess loss of fluid in the feces. In some diarrhoea, the secretory component is predominated, and other diarrhea is characterized by hypermotility 2 . Different plant extracts usually mediate their spasmolytic action through blockage of Ca2+ influx. This indicates that the plant extract is preventing the increase of calcium ions and muscle tension 3 . This result is in agreement with the results of others which examined the effects of other plants on isolated smooth muscle contraction. The essential oil in current analysis characterized by the domination of substances: Myristicin apiol, a-pinene, b-pinene 8 . Flavonoids secondary plant metabolites occurring widely in vegetables have been shown to display a remarkable array of biochemical and pharmacology actions, including relaxing effect on intestinal smooth muscles 19 . There have been a number of reports that plants containing essential oil 8 and containing flavonoids 4 possess inhibitory effects on smooth muscles activity. There have been several reports that the antispasmodic effect of the extract is associated with the presence of Flavonoids and essential oil of the plants. Some of them inhibit intestinal motility in vitro. Quercetin, exhibited antispasmodic effect, on guinea-pig isolated ileum. Apigenin 5 and luteolin inhibited the contractions of the isolated intestine. Also essential oil, such as a-pinene, b-pinene exhibited relaxation effect on isolated rat ileum contractions 19 . These materials have been reported to exhibit calcium antagonist and anticholinergic activities 3 . Several studies in vitro and in vivo showed that those compounds can regulate disturbances of the gastrointestinal tract. Their effects depend on the mechanism involved and the tissue. The spasmolytic effect may due to blockade of the calcium channel. And these flavonoids 4 and essential oil have been reportedly present in the extract of Parsley 5 , therefore, the extract of Parsley activity possibly was due to this substances.
Other studies reported that ethanol extract of Calotropis procera leaves produced an excellent spasmolytic effect on gastrointestinal smooth muscles (guinea pig ileum) probably due to flavonoids of the crude extract 23 .
In another set of experiments, the extract , s mechanism of actions and the role of histamine (general) agonist and Cimetidine (antagonist H2 receptor) on relaxation was investigated. To evaluate whether the extract has relaxed intestine by binding to agonist and antagonist H2 histaminergic receptors, the relaxing effect of the extract was examined in the presence of Histamine and Cimetidine. The ineffectiveness of both the spasmolytic effect of extract indicates that agonist (general) and antagonist H2 histaminergic receptors are not involved in this action. The relaxation of smooth muscles was induced by different mechanisms that include the blocking action on excitatory/inhibitory pathways such as cholinergic 24 , histaminergic 25 , adrenergic 19 , purinergic 26 , GABAergic 9 .
One of the possible mechanisms for the spasmolytic activity of the extract could be mediated through the inhibition of histaminic receptors 3 .

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In the alimentary tract, it is well-known that cholinergic and histaminergic transmissions are involved in the regulation of intestinal motility. Drugs can influence cholinergic or histaminergic transmission by acting on their receptors or by affecting the release of endogenous acetylcholine or histamine, respectively. These results show that histamine can induce contractions in isolated chicken ileum 27 and in the guinea pig ileum 25 .
Other studies also reported that histamine produced dose-dependent contractions in rectum and colon of rat 28  Histamine exerts its actions by binding to four GPCRs that are differentially expressed throughout the body and designated as the H1, H2, H3 and H4 receptors 30 .
Histamine induces production of inositol phosphates in smooth muscles via G aq protein activation 13 . Histamine was used as an agonist for stimulating ileum smooth muscles. Histamine is known to cause contraction of smooth muscles of the gastrointestinal tract. Application of histamine to intact smooth muscles produced a concentrationdependent membrane depolarization and increased excitability. On the other hand, Histamine as a histaminergic receptor agonist 3 activates receptoroperated Ca2+ channel, elevates [Ca2+] and induces contraction via H1 receptors 17 .
Histamine also activates phospholipase C and enhances inositol triphosphate (IP3) production 13 followed by the promotion of Ca2+ release from intracellular Ca2+ pools (sarcoplasmic reticulum).
Histaminergic control of gastrointestinal contractility and motility is complex and involves all histamine receptor subtypes. H1 receptors, located in smooth muscle cells, contribute to contractility by increasing calcium availability at the sarcoplasmic level 30 . Histamine-induced contraction of guinea pig ileum is mediated by H1receptors. H2R is coupled both to adenylate cyclase and to phosphoinositide second messenger systems via separate GTP-dependent mechanisms. Receptor binding stimulates activation of c-Fos, c-Jun, Protein Kinase C (PKC) and p70S6kinase 16 .
Histamine provoked contraction and membrane depolarization in the longitudinal smooth muscle cells of the guinea pig ileum. This may have been the result of on increasing ca2+ partition through voltage-gated and receptor-operated ca2+ channels 17 . There is convincing evidence that can be significant for variation in the degree of the partition of extracellular and intracellular ca2+ in smooth muscles contraction. Calmodulin (Cam) plays an important role in contraction behaviour and electrical rhythmicity 8 .
It's evident that histamine stimulates the enteric neurons and H2 agonists shown the increase, the amplitude of electrically evoked contractions of the longitudinal muscle. Therefore it is possible that histamine activates the longitudinal muscle excitatory motor neurons. Histamine produces a contraction of the circular muscle in the guinea pig small intestine. Histamine, acting via H1-and H2receptors makes the depolarization of guinea pig myenteric neurons with the increase in the input resistance, augmented excitability and repetitive spike demonstrated. The enzymes involved in the synthesis of histamine and its mRNA is present in enteric neurons. Histamine plays a functional role in intestinal motility 29 . The histamine H2 receptor is a couple to the adenylate cyclase system in a variety of tissues (e.g., brain, stomach, heart, gastric, mucosa, lung). Moreover, cell lines transfected with the cloned H2 receptor genes showed an H2 receptor-mediated increase of cAMP. In addition, alternative signaling pathways for H2 receptor have been identified. An H2 receptor-mediate increase of the intercellular ca 2+ concentration and/or IP 3 level was observed.
The distinctive finding in this study is that an alcohol extract of parsley seeds is a relaxant of rat ' s ileum smooth muscles and that histaminergic H2 receptors antagonist (Cimetidine) and agonist (Histamine) do not change relaxation induced by the extract. And cimetidine did not significantly affect the activity of the extract, suggesting that extract does not have any effects on histaminergic H2 receptors. It may be used to treat digestive disorders like cramps and diarrhea. Other experimental approaches will be necessary to delineate the exact relationship between extract of parsley and the histaminergic receptors.

Conflict of Interest Statement
We declare that we have no conflict of interest 6. Acknowledgment