Antidiarrhoeal and antispasmodic activity of leaves of Syzygium cumini L . ( Myrtaceae ) mediated through calcium channel blockage

Syzygium cumini L. Skeels (Myrtaceae) commonly known as jambolan is used as traditional medicine to treat gastrointestinal disorders in children in Brazil. This work is one of the first to evaluate the antidiarrhoeal and antispasmodic activity of the standardized extract of S. cumini leaves (HESc) in experimental models in vitro and in vivo rodents. Mice pre-treated with HESc (100, 250 and 1000 mg/kg) and atropine (1.0 mg/kg) had reduced intestinal transit velocity of 11.0; 23.2 and19.1%, respectively compared to saline control (46.6±0.9). In isolated rats jejunum, HESc (50, 150 and 300 μg/mL) shifted to the right cumulative concentration-response curves to ACh with changing maximum effect (Emax), which is characteristic of non-competitive antagonism to ACh. HESc also promoted relaxation (Emax 90.2±5.8%) in preparations pre-contacted with KCl (75 mM). Additionally, it reduced the maximal CaCl2induced response in 15.4; 56.3 and 92.1% in a concentration-dependent manner. The study results show that HESc has an antidiarrhoeal and spasmolytic potential that can be partly explained by the reduction of intestinal transit velocity and blockage of the voltage-dependent calcium channels in the smooth intestinal muscle.


INTRODUCTION
Diarrhoea can be defined as a symptom of the gastrointestinal disorder; it is characterized by increase in stool frequency and alteration in consistency.It results from the imbalance between the absorptive and secretory mechanisms in the intestinal tract accompanied by hypermotility, bringing about excess loss of body fluids and electrolytes in feces (Sharma et al., 2015;Fernández-Bañares et al., 2015;Nemeth and Pfleghaar, 2017).
Treatment of gastrointestinal disorders and the search for new therapeutic agents are still a challenge.A potential antidiarrhoeal agent may exhibit its effect by inhibiting the gut motility (spasmolytic) and/or electrolyte outflux in the form of wet droppings, for example.The World Health Organization (WHO) has approved the use of traditional (folklore) medicines for treating many *Corresponding author.E-mail: fabiodesouza8@gmail.com.
Natural products continue to play a highly significant role in drug discovery and development process.When analyzing drugs approved by Food and Drug Administration (FDA) between 1981 and 2014, approximately half of the drugs were based on natural products or derivatives thereof (Newman, 2013;Lahlou, 2013;Harvey et al., 2015;Newman et al., 2016).
Medicinal plants till date are quite used to treat various diseases.For example, Syzygium cumini is extensively used for the treatment of different diseases, such as inflammation, constipation, diarrhoea, obesity, urinary disorders, diabetes and hypertension.Despite this extensive use in folk medicine, most of the time there are no scientific studies that prove people use them (Zahoor et al., 2017;Mukherjee et al., 2017;Baliga et al., 2011;Ayyanar and Subash-Babu 2012).
Syzygium cumini L. Skeels, of the Myrtaceae family, popularly known as jamun, is included in the List of Medicinal Plants of Interest to the Public Health System (Renisus), issued by the Ministry of Health, which includes plants with potential use for medicines (Souza, 2005;Brasil, 2009;Swami, 2012;Subash-Babu and Ayyanar et al., 2013).All parts of S. cumini are widely used as traditional medicine, for example, juice of tender leaves is given in goat milk to treat diarrhoea in children (Morton, 1963;Corrêa, 1974;Lainetti and Brito, 1979;Nadkarni, 1976).
The antidiarrhoeal activity was investigated using extracts obtained from the stem bark and seeds of S. cumini (Mukherjee et al., 1998;Mazumder et al., 2006;Shamkuwar et al., 2012;Chandra, 2013).However, there is no report available on the hydro-alcoholic extract of the leaves of S. cumini on its anti-diarrhoeal or antispasmodic activity despite its medicinal use in diarrhoea.
The research group previously showed that a standardized hydroalcoholic extract prepared from the leaves of S. cumini (HESc) is safe, with no evidence of toxicity in rodents (Silva et al., 2012).We subsequently demonstrated the hypotensive and antihypertensive activity of HESc, causing a reduction in vascular reactivity associated with the inhibition of extracellular calcium influx, whose mechanism was attributed to the marked presence of flavonoids (Mahmoud et al., 2001;Ribeiro et al., 2014).This work is the first to evaluate HESc in experimental models in vitro and in vivo animals, analyzing the intestinal transit velocity in Swiss albino mice, and the contractile activity in isolated rat jejunum, respectively.

Preparation of crude extract
The leaves were mechanically ground to give 920 mg powder.This was added to 1 L of ethanol (70%) and mixed at 8 h each for 72 h.After this period the hydroalcoholic extract was filtered using a cotton funnel.After this process, the extract was concentrated using a rotatory evaporator under reduced pressure and filtered again.We obtained a concentrate of 150 mg/ml and a yield of 16.3% proportional to the 920 mg initially obtained.Such concentrate was denominated in a hydroalcoholic extract of S. cumini (HESc).Finally, the extract was lyophilized to obtain a powder and the dry residue obtained was solubilized in distilled water to a concentration of 10 mg/ml.It was re-diluted in distilled water as needed for each experimental protocol.

Experimental animals
Swiss albino mice (25 to 30 g) and Wistar albino rats (250 to 300 g) of either sex from the Universidade Federal de São Luis, Brazil were used.Animals were housed under controlled temperature (25±1°C) and lighting (lights on 06:00 to 18:00 h); they had free access to food and potable water.All procedures described in the present study were approved by the Animal Research Ethics Committee of the State University of Maranhão, Brazil (Protocol number 003584/2014-97).

Small intestinal transit
Swiss albino mice were fasted for 6 h prior to the experiments, but were allowed free access to water.The animals were treated with HESc (100, 250 or 1000 mg/kg, p.o., respectively), atropine sulfate (1.0 mg/kg, p.o.) or saline (10 mL/kg, p.o. n=6), 60 min prior to the administration of a 5% charcoal suspension in 1% guar gum (0.1 mL/10 g body weight, p.o.).After 30 min, the animals were euthanized and their small intestines were removed.The distance traveled by the charcoal plug from the pylorus to the cecum was measured and expressed as a percentage of the total intestinal length (adapted from Freire et al., 2011).

Effect of HESc on ACh-induced cumulative dose-response curves in isolated rat jejunum
All rats were euthanized by decapitation with guillotine following the principles of laboratory animal care based on the guidelines of the bioethics committee.Segments of jejunum (1.5 cm long) were suspended in a 10 mL organ bath containing Tyrode's solution (composition in mmol/L: NaCl, 137; KCl, 2.7, MgCl2 • 6H2O, 0.5; CaCl2 • 2H2O, 1.8; NaH2PO4, 0.4; NaHCO3, 12; glucose, 5.5), aerated with 95% O2, 5% CO2 (pH 7.4) and maintained at 37°C.The preparations were set up under a tension of 1 g and responses were recorded on a smoked Kymograph paper through an isotonic frontal writing lever (magnification x 6).After 30 min equilibration period, cumulative concentration-response curves for ACh (10 -9 to 10 -4 M) were recorded in the absence and presence of HESc (50, 150 and 300 µg/mL).This curve was compared with those obtained in the absence of HESc and the results were expressed as percentages of the maximal response to ACh alone (Van Rossum, 1963).

Effect of HESc on KCl-induced tonic contractions in isolated rat jejunum
Isolated rat jejunum was obtained as described earlier.Segments of jejunum (1.0 cm long) were suspended in a 5 mL organ bath containing Tyrode's solution aerated with 95% O2, 5% CO2 (pH 7.4) and maintained at 37°C.The preparations were set up under a tension of 1 g.In addition, for the recording of the isometric tension, the thread from the muscle strips was attached to an isometric force transducer that was connected to a bridge amplifier (ADInstruments Ltd, Grove House, Hastings, U.K.).Isometric tension changes were digitized using either PowerLab/4SP (ADInstruments Ltd, Grove House, Hastings, U.K.) and stored on a personal computer for later analysis.After 30 min equilibration period, segments of jejunum were contracted with KCl (75 mM) and when a stable contraction was attained (15-20 min), HESc (9; 27; 81; 243 and 729 µg/mL) was cumulatively added in an attempt to obtain dose-relaxation curves.The relaxant effect induced by HESc was expressed as the reverse percentage of the initial contraction force elicited by KCl.

Effect of HESc on CaCl2-induced cumulative dose-response curves in calcium-free solution in isolated rat jejunum
The jejunum was mounted as described earlier.The preparations were set up under a tension of 1 g, and responses were recorded on a smoked Kymograph paper through an isotonic frontal writing lever (magnification x 6).After the stabilization during 30 min in normal Tyrode's solution, the external calcium was eliminated with depolarizing Tyrode's solution (KCl, 70 mM; Ca 2+ -free).Cumulative concentration-response curves of Ca 2+ were obtained by cumulatively adding CaCl2 (3 x 10 -5 to 10 -1 M) in the absence and presence of HESc (27, 81 and 243 µg/mL), which were added to the bath 10 min before addition of Ca 2+ .This curve was compared with those obtained in the absence of HESc and the results were expressed as percentages of the maximal response to CaCl2 alone (Van Rossum, 1963).

Statistical analysis
Values were expressed as mean ± S.E.M. Statistical analysis was performed using GraphPad Prism 5.01 software (GraphPad Software Inc., San Diego, CA, USA).Differences between means were compared using t-test (non-paired) and one-way ANOVA followed by Bonferroni's test as appropriate and p values < 0.05 were considered indicative of significance.

RESULTS AND DISCUSSION
In this study, it is shown for the first time the investigation of the antidiarrhoeal and antispasmodic activities of the Monteiro et al. 13 HESc, analyzing the intestinal transit velocity in Swiss albino mice, and the contractile activity in isolated rat jejunum, respectively.It is demonstrated that HESc has antidiarrhoeal and antispasmodic effect by decreasing the speed of intestinal transit and blocking the influx of calcium, respectively.Charcoal meal test in mice is a method used to study the effect of drugs on the motility of intestine (Misar, 2000).The transit velocity of the small intestine compared to the effect produced by saline control (46.6±0.9%), was reduced to 11.0; 23.2; 19.1 and 38.5%, respectively, after pre-treatment of mice with HESc (100, 250 and 1000 mg/kg) and atropine (1.0 mg/kg), administered orally, 60 min before administration of coal meal.Although HESc was less efficient in decreasing intestinal transit compared to atropine (standard drug), it reduced intestinal transit significantly in all doses tested compared to the control as shown in Figure 1.
Anti-diarrhoeal activity has already been demonstrated in studies carried out with the seeds and bark of the stem of S. cumini.In both studies, it was possible to show a significant reduction in gastrointestinal motility in tests of coal meal in rats.The underlying mechanism of action of the plant extract appeared to be antispasmodic whereby the extract produced relief from diarrhea (Mukherjee et al., 1998;Shamkuwar et al., 2012;Srivastava and Chandra, 2013).
It is well known that aqueous herbal medicines are traditionally used for their antispasmodic and antidiarrhoeal activity in various countries (Hajhashemi et al., 2000;Mujumdar et al., 2000;Sadraei et al., 2003).Non-specific anti-diarrhoeal drugs involve actions on intestinal transit that results in symptomatic improvement in a variety of conditions.Furthermore, the study on antispasmodic effect might help to deduce the possible mechanism of action (Schiller, 1995).
Antispasmodics may be classified, for example, into antimuscarinics, smooth muscle relaxants (that is, drugs that directly inhibit smooth muscle contractility, for example, by increasing cyclic AMP levels or by interfering with the intracellular calcium pool), similar agents papaverine and Ca 2+ blocking channels (especially Ca 2+ L-channel blockers) (Christen, 1990;Singh et al., 2003).Muscarinic receptors of the M 3 subtype are present in the intestinal smooth muscle.These receptors are responsible for initiating contraction in response to acetylcholine (ACh) binding.This neurotransmitter is released by parasympathetic postganglionic neurons that innervate the digestive tract (Weiser et al., 1997).Upon binding with ACh, the M 3 receptor initiates a cellular signaling cascade.Briefly, the alpha subunit of the Gq/11 protein activates the effector phospholipase C (PLC), which increases the inositol triphosphate (IP 3 ) secondary messenger responsible for releasing calcium from the sarcoplasmic reticulum.This Ca 2+ release activates voltage-gated Ca 2+ (Ca V ) channels indirectly that leads to influx of Ca 2+ from extracellular fluid (Caulfield, 1993; Eglen et al., 1996;Honda et al.,1996;Catterall et al, 2005).The isolated rat jejunum was used in this study to initially investigate whether the reduction of intestinal motility caused by HESc was mediated by competitive antagonism to the M 3 receptor, with consequent interference in the availability of intracellular Ca 2+ .For this, the effect of HESc on cumulative concentrationresponse curves to the addition of ACh ( 10-9 to 10 -4 M) was evaluated.The pD 2 value (pD 2 = -log EC 50 , negative logarithm of molar concentration of agonist that caused half-maximal response) was 6.4±0.05M. In the presence of HESc (50, 150 and 300 µg/mL), the pD 2 value was altered to 5.8±0.2,6.2±0.2 and 5.1±0.1 M, respectively, and E max to ACh was reduced in 17.8, 34.3 and 57.3% (Figure 2), suggesting a non-competitive type antagonism.
Spasm is characterized by a muscle contraction and in the smooth muscle this contraction occurs after the elevation of the intracellular calcium concentration ([Ca  2+ ] i ) due to the opening of the voltage-dependent calcium channels (Ca V ) present in the plasma membrane or due to its release of sarcoplasmic reticulum (RS) controlled by secondary messengers, for example, IP 3 .The functional regulation of [Ca  2+ ] i to trigger a contractile response in smooth muscle is related to two stimuli that lead to two types of couplings: (1) electromechanical coupling, which is involved with the membrane potential change (Vm) and (2) drug-mechanical coupling when the contraction induced by an agonist is always greater than that observed only with the change of Vm (Al-Zuhair et al., 1996;Rembold, 1996;Bolton, 1979).
The contraction in the smooth muscle in response to several agents is often composed of two phases: a fast and unstained phasic component, followed by a slow and sustained tonic component (Breemen and Saida, 1989).The phasic component is due in part to the Ca 2+ of the sarcoplasmic reticulum and the tonic component is mainly due to Ca 2+ from the extracellular medium entering the cell through the Ca V (Abdellatif, 1989;Kobayashi et al., 1989;Takano and Kamiya, 1996).
In order to verify whether HESc would promote relaxation of the pre-contracted jejunum, which would be suggestive, at a functional level, of blocking Ca 2+ influx through the plasma membrane, its effects were evaluated on the tonic component of the contractile response induced by KCl 75 mM (electromechanical coupling).It was observed that HESc promotes relaxation of the jejunum (Figure 3B) in a concentration-dependent manner with a E max of 90.2±5.8%(Figure 4); we hypothesized that HESc would prevent Ca 2+ influx through Ca V .
To confirm this hypothesis, the effect of HESc on the cumulative concentration-response curves to the CaCl 2 was evaluated.The jejunum was contracted with increasing concentrations of CaCl 2 (3 x 10 -5 M to 10 -1 M), with a pD 2 value of 2.07±0.09M. In the presence of HESc, at 27, 81 and 243 µg/mL, the pD 2 values were lowered to 1.59±0.12,1.83± 0.14 and 1.29±0.06M, respectively.In addition, HESc displaced the cumulative    concentration-response curves of CaCl 2 to the right, in a non-parallel manner and the maximal response was reduced, in a concentration-dependent manner, by 15.4; 56.3 and 92.1%, respectively (Figure 5), suggesting that the antispasmodic effect is possibly mediated through the inhibition of Ca 2+ influx probably through of Ca V .Other studies show that antispasmodic constituents present in various medicinal plants mediate their effect generally by blocking the calcium channel (Ghayur et al., 2006;Gilani et al., 2006;Shah et al., 2010).Similar results of inhibition of contractile responses to calcium were found in previous studies with the hydroalcoholic extract of S. cumini by our laboratory in preparation of vascular arteries rings isolated from normotensive and spontaneously hypertensive rats.The effects were attributed to the presence of flavonoids detected by phytochemical screening (Abreu et al., 2002;Ribeiro et al., 2014)

Conclusion
In this study, it can be concluded that the antidiarrhoeal effect of HESc, observed by the reduction of the intestinal transit, can be explained by the blockage of calcium influx through Ca V responsible for the antispasmodic activity.These properties may explain the use of S. cumini as an antidiarrhoeal agent in traditional medicine and contribute to the future indication of S. cumini as a possible therapeutic alternative to treat gastrointestinal diseases.However, further studies are needed to explore the secondary metabolites responsible for the results obtained in this study.