GC–MS Analysis and In Vivo and Ex Vivo Antidiarrheal and Antispasmodic Effects of the Methanolic Extract of Acacia nilotica

This present study evaluated and rationalized the medicinal use of the fruit part of Acacia nilotica methanolic extract. The phytochemicals were detected using gas chromatography–mass spectrometry (GC–MS) while the in vivo antidiarrheal test was done using Swiss albino mice. To determine the details of the mechanism(s) involved in the antispasmodic effect, isolated rat ileum was chosen using different ex vivo assays by maintaining a physiological environment. GC–MS results showed that A. nilotica contained pyrogallol as the major polyphenol present (64.04%) in addition to polysaccharides, polyphenol, amino acid, steroids, fatty acid esters, and triterpenoids. In the antidiarrheal experiment, A. nilotica inhibited diarrheal episodes in mice significantly (p < 0.05) by 40% protection of mice at 200 mg/kg, while 80% protection was observed at 400 mg/kg by the orally administered extract. The highest antidiarrheal effect was observed with loperamide (p < 0.01), used as a control drug. In the ex vivo experiments, A. nilotica inhibited completely in increasing concentrations (0.3 to 10 mg/mL) the carbachol (CCh; 1 µM) and high K+ (80 mM)-evoked spasms in ileum tissues at equal potencies (p > 0.05), similar to papaverine, a dual inhibitor of the phosphodiesterase enzyme (PDE) and Ca++ channels. The dual inhibitory-like effects of A. nilotica on PDE and Ca++ were further validated when A. nilotica extract (1 and 3 mg/mL)-pre-incubated ileum tissues potentiated and shifted isoprenaline relaxation curves towards lower doses (leftward), similar to papaverine, thus confirming the PDE inhibitory-like mechanism whereas its CCB-like effect of the extract was confirmed at 3 and 5 mg/mL by non-specific inhibition of CaCl2-mediated concentration response curves towards the right with suppression of the maximum peaks, similar to verapamil, used as standard CCB. Thus, this study characterized the chemical composition and provides mechanistic support for medicinal use of A. nilotica in diarrheal and hyperactive gut motility disorders.


Introduction
Gastrointestinal (GI) motility plays an important role in digestive and absorptive processes of the gut, essential for pushing intestinal material, mixing this with digestive juices, and preparing undigested foods for excretion. Diarrhea, characterized by an increased frequency of bowel movements, wet stool, and abdominal cramps, is a serious health problem [1,2]. Diarrhea can be caused by several factors, such as infections, food intolerance, intestinal disorders, etc. [3][4][5], and might be a symptom of many other ailments, including IBS and diabetes [6,7]. Gut motility is controlled via various physiological agents, such as, acetylcholine (ACh), prostaglandin E2, serotonin (5-hydroxytryptamine or 5-HT),

Animals
From the Animal Care Unit, 'College of Pharmacy, Prince Sattam bin Abdulaziz University, Saudi Arabia', Swiss albino mice (25-30 g) were obtained for in vivo studies and rats (200-250 g) for ex vivo experiments and were kept at a temperature optimum (22 • C), relative humidity (55%), and exposure to a light/dark cycle. All animals were fed a regular diet of pellets and had unrestricted access to water. Prior to the ex vivo studies, mice fasted for 24 h, and cervical dislocation was performed under light sedation, with death confirmed by elimination of ear reflexes. All experiments (in vivo and ex vivo) were carried out with caution and in accordance with the guidelines outlined in the NRC [32]. The Bio-Ethical Research Committee (BERC) at 'Prince Sattam Bin Abdulaziz University' approved the study protocol with the approval number BERC-004-12-19.

GC-MS Analysis
The phytochemical investigation of the methanolic extract of A. nilotica was per-formed by GC-MS to detect the presence of several phytoconstituents. The chromatographic separation of metabolites was carried on a capillary column 60 M TRX 5-MS (30 m × 250 µm I.D. 0.25 µm film) using 2 µL of sample injection volume. The oven temperature program was as follows: 80 • C initially for 3 min and then ramped at a rate of 10 • C/min to 280 • C for 19 min. The carrier gas was set at a constant flow rate of 1.21 mL/min. The injection port, transfer line, and ion source were set to 260 • C, and the mass-scanning range was set to 40 to 650 m/z in scan mode. The injection was executed in split mode with a 10:1 split ratio, and a 3-min solvent delay time was set for the samples. Identification of individual phytoconstituents was achieved using National Institute of Standards and Technology (NIST) libraries and the mass spectra of literature [30,33].

In Vivo Antidiarrheal Study
Twenty mice were divided into five groups, each with an equal number of mice, at random. Mice in the first group were administered an oral gavage of saline (10 mL/kg) after a twenty-four-hour fast and were labeled as the negative control group. The second and third groups (test groups) were given two increasing doses of A. nilotica methanolic extract, 200 and 400 mg/kg, respectively, after a pilot screening for dose selection. As a positive control, the fourth group of mice was administered loperamide (10 mg/kg). Each animal was kept in the cage, with a blotting sheet on the floor to allow a blind observer to determine the presence or absence of diarrhea. All mice were given castor oil (10 mL/kg) orally after an hour. All blotting sheets from individual cages were checked for typical diarrheal droppings after 4 h. If no diarrheal spots were noticed on the blotting sheet, protection was documented [34,35].

Ex Vivo Experiments on Isolated Rat Ileum
A previously documented approach was used to sacrifice rats and to separate the final part of the small intestine (ileum) [36]. Ileum tissues (2-3 cm) were cleaned from neighboring tissues and luminal feces and mounted in an isolated organ bath (emkaBATH, Paris, France) attached to transducer and IOX software. The temperature was set to 37 • C, and a freshly prepared Tyrode's solution bubbled with carbogen gas was provided as a physiological medium in the tissue baths (20 mL The tissues were stabilized for 30 min with the addition of acetylcholine (0.3 M) at regular intervals (5 min) while 1 g tension was applied by clockwise rotation of the transducer knob. CCh and high K + (80 mM) were employed to induce prolonged contractions after stabilization, and A. nilotica was added to the bath solution in increasing concentrations until the maximal and/or complete relaxation of tissue was achieved. The inhibitory effect of A. nilotica on CCh and K + -mediated contractions was observed, which could indicate pharmacodynamics such as voltage-gated Ca ++ channel blockade and/or PDE inhibition. Multiple smooth muscles are depolarized by K + (>30 mM), which activates Ca ++ channels (L-type), resulting in prolonged contractions [37]. PDE-inhibitors, on the other hand, are agents that, at comparable concentrations, reverse CCh and high K + -mediated contractions, whereas verapamil (CCB) shows significantly higher potency against high K + compared to CCh-mediated contractions [38].

Ca ++ Inhibitory Confirmation
After the observation of preliminary relaxation of A. nilotica against high K + , ileum tissues were incubated in Ca ++ -free Tyrode's solution with EDTA (0.1 mM) for 45 min to confirm Ca ++ channel blocking (CCB). A Ca ++ -free solution was replaced with a K + -rich and Ca ++ -free Tyrode's solution at the following concentrations (mM): KCl 50, NaCl 91.04, MgCl 2 1.05, NaHCO 3 11.90, NaH 2 PO 4 0.42, glucose 5.55, and EDTA 0.1. After 45 min of incubation in this solution in the presence and absence of increasing concentrations of A. nilotica, CaCl 2 CRCs were produced, and the findings were compared to the standard CCB agent, verapamil [39].

PDE Inhibitory Confirmation
The relaxing effect of A. nilotica against high K + and CCh at identical concentrations is an indication of PDE inhibition [40]; therefore, dose-mediated inhibitory curves of isoprenaline against CCh in the presence and absence of A. nilotica were used to indirectly validate PDE inhibition. PDE blockage was indicated by the potentiation of isoprenaline curves to the left, similar to papaverine, a typical PDE inhibitor, utilized as a control [41].

Statistical Analysis
The statistical analyses were performed as the mean ± standard error of the mean (SEM), with "n" being the number of experiments that were repeated. The median effective concentrations (EC 50 ) are geometric means with 95% confidence intervals (CIs). The statistical criteria utilized for multiple comparisons of concentration-response curves (CRCs) with controls were Student's t-test or two-way ANOVA followed by Bonferroni's post-test. W the Chi-square (χ 2 ) test, all groups were statistically compared to a saline control group for diarrhea protection. p < 0.05 was regarded as statistically significant. For CRC regression analysis, Graph Pad Prism (version 4) was used.

In Vivo Antidiarrheal Effect
In comparison to the saline group, both increasing orally delivered dosages of A. nilotica in mice showed significant antidiarrheal effects (Table 2). At the lower tested dose of 200 mg/kg, two out of five mice showed protection, suggesting 40% protection, whereas the higher dose of 400 mg/kg demonstrated 80% protection from diarrhea. In all five cages of mice treated with loperamide (10 mg/kg), no diarrheal spot was observed (100% protection), as detailed in Table 2. Table 2. Antidiarrheal activity of the methanolic extract of A. nilotica on castor oil (10 mL/kg)-induced diarrhea in mice.

In Vivo Antidiarrheal Effect
In comparison to the saline group, both increasing orally delivered dosages of A. nilotica in mice showed significant antidiarrheal effects (Table 2). At the lower tested dose of 200 mg/kg, two out of five mice showed protection, suggesting 40% protection, whereas the higher dose of 400 mg/kg demonstrated 80% protection from diarrhea. In all five cages of mice treated with loperamide (10 mg/kg), no diarrheal spot was observed (100% protection), as detailed in Table 2. Table 2. Antidiarrheal activity of the methanolic extract of A. nilotica on castor oil (10 mL/kg)-induced diarrhea in mice.

Calcium Channel Blocking (CCB)-like Effect
To confirm the Ca ++ inhibitory activity, preincubation of ileum tissues with A. nilotica methanolic extract skewed the Ca ++ CRCs curves at tested dosages of 3 and 5 mg/mL ( Figure 4A) towards the right with suppression of the maximum effect. Similarly, verapamil and papaverine, at respective preincubated concentrations (0.01 and 0.03 µM; verapamil) and (1 and 3 µM; papaverine), also deflected Ca ++ curves towards the right with suppression of the highest peaks as shown in Figure 4B,C.
To confirm the Ca ++ inhibitory activity, preincubation of ileum tissues with A. nilotica methanolic extract skewed the Ca ++ CRCs curves at tested dosages of 3 and 5 mg/mL ( Figure 4A) towards the right with suppression of the maximum effect. Similarly, verapamil and papaverine, at respective preincubated concentrations (0.01 and 0.03 µM; verapamil) and (1 and 3 µM; papaverine), also deflected Ca ++ curves towards the right with suppression of the highest peaks as shown in Figure 4B,C.

Discussion
To provide the basis to the traditional medicinal report of A. nilotica in diarrhea and gut spasms [42], the methanolic extract of A. nilotica was evaluated scientifically using rodents while its phytochemical analysis was determined by GC-MS. In vivo, A. nilotica was examined in a castor oil-evoked diarrhea model and found to have dose-mediated antidiarrheal action by preventing the characteristic diarrheal drops as compared to the saline control group. Castor oil is known to increase intestinal fluids, causing diarrhea indirectly through the formation of recinoleic acid, which ultimately alters the electrolytes and water transport and elicits excitations in transverse and distal segments of the colon [43]. Similar to the positive control drug, loperamide, a popular antidiarrheal treatment [44], pre-administration of A. nilotica protected mice from diarrhea in a dose-dependent manner. The methanolic extract of A. nilotica was examined at cumulative doses in isolated rat ileum to determine the possible pharmacodynamics involved in the observed antidiarrheal activity [45]. Based on earlier results that antispasmodic drugs mediate gut inhibitory effects via Ca ++ channel blocking [44] and/or PDE inhibition [46], we evaluated A. nilotica extract on the evoked contractions in rat ileum by CCh and high K + [47]. The EC50 values obtained from both types of inhibitory curves of A. nilotica against CCh and high K + showed no statistical difference (p > 0.05). Similarly, papaverine, a dual Ca ++ channel and PDE inhibitor [38], suppressed both CCh and high K + -evoked spasms at comparable concentrations, but verapamil, a typical CCB [39,48], selectively inhibited high K + at a lower concentration compared to CCh. This indicates that, similar to papaverine, A. nilotica has dual inhibitory mechanisms for PDE inhibition and Ca ++ channels. PDE-inhibitors, which block PDE, result in a cAMP increase in tissues and thus cause relaxation. PDE hinders smooth muscle relaxation by converting cAMP into its inactive form (AMP) [49]. Hence, A. nilotica was evaluated indirectly for PDE-inhibition and cAMP elevation by constructing isoprenaline-induced inhibitory CRCs in the absence and presence of pre-incubated tissues with the test substance. In pre-incubated ileum tissues of A. nilotica, potentiation of isoprenaline's inhibitory CRCs towards lower dosages (leftward) verified its PDE-inhibitory character, and the results were equivalent to

Discussion
To provide the basis to the traditional medicinal report of A. nilotica in diarrhea and gut spasms [42], the methanolic extract of A. nilotica was evaluated scientifically using rodents while its phytochemical analysis was determined by GC-MS. In vivo, A. nilotica was examined in a castor oil-evoked diarrhea model and found to have dose-mediated antidiarrheal action by preventing the characteristic diarrheal drops as compared to the saline control group. Castor oil is known to increase intestinal fluids, causing diarrhea indirectly through the formation of recinoleic acid, which ultimately alters the electrolytes and water transport and elicits excitations in transverse and distal segments of the colon [43]. Similar to the positive control drug, loperamide, a popular antidiarrheal treatment [44], pre-administration of A. nilotica protected mice from diarrhea in a dose-dependent manner. The methanolic extract of A. nilotica was examined at cumulative doses in isolated rat ileum to determine the possible pharmacodynamics involved in the observed antidiarrheal activity [45]. Based on earlier results that antispasmodic drugs mediate gut inhibitory effects via Ca ++ channel blocking [44] and/or PDE inhibition [46], we evaluated A. nilotica extract on the evoked contractions in rat ileum by CCh and high K + [47]. The EC 50 values obtained from both types of inhibitory curves of A. nilotica against CCh and high K + showed no statistical difference (p > 0.05). Similarly, papaverine, a dual Ca ++ channel and PDE inhibitor [38], suppressed both CCh and high K + -evoked spasms at comparable concentrations, but verapamil, a typical CCB [39,48], selectively inhibited high K + at a lower concentration compared to CCh. This indicates that, similar to papaverine, A. nilotica has dual inhibitory mechanisms for PDE inhibition and Ca ++ channels. PDE-inhibitors, which block PDE, result in a cAMP increase in tissues and thus cause relaxation. PDE hinders smooth muscle relaxation by converting cAMP into its inactive form (AMP) [49]. Hence, A. nilotica was evaluated indirectly for PDE-inhibition and cAMP elevation by constructing isoprenaline-induced inhibitory CRCs in the absence and presence of preincubated tissues with the test substance. In pre-incubated ileum tissues of A. nilotica, potentiation of isoprenaline's inhibitory CRCs towards lower dosages (leftward) verified its PDE-inhibitory character, and the results were equivalent to papaverine, a known PDEinhibitor [50]. CCh-mediated smooth muscle spasm is well recognized to be inhibited by PDE inhibitors [51]. In order to explore the possibility of additional antispasmodic mechanisms in A. nilotica extract, it was tested for Ca ++ ion inhibitory effect.
Substances that reverse high K + (>30 mM)-mediated spasm are considered as CCBs [52], hence to support and confirm further the CCB-like action of A. nilotica, in previously Ca ++free tissues, the ileum tissues were preincubated with A. nilotica at increasing concentrations. Ca ++ -CRCs were made in the absence of A. nilotica and pre-incubated tissues with A. nilotica, which repelled Ca ++ -CRCs to the right with suppression of the maximum peak, similar to papaverine, a dual inhibitor of PDE and Ca ++ channels. The plant Ca ++ -CRC comparison with verapamil, a standard CCB [39], further confirmed the additional CCB-like mechanism of A. nilotica. Previously published findings of the antispasmodic effect of A. nilotica pods in rabbit jejunum support this CCB-like effect [42]. Polysaccharides, polyphenols, amino acids, steroids, fatty acid esters, and triterpenoids were found in the GC-MS analysis of the A. nilotica methanolic extract. Pyrogallol was discovered to be one of the major phytoconstituents of A. nilotica; it is a polyphenol that is present in (64.04%) the extract and has antibacterial activity [53], whereas 4-O methylmannose is present in the second highest concentration (17.72%); this is a polysaccharide that has been reported to have anti-alopecic, anti-cirrhotic, anti-neuropathic, cholesterolytic, lipotropic, and sweetening properties [54]. In COPD patients, N, N-dimethylglycine may be useful as a diagnostic of protein degradation.

Conclusions
These findings characterized the chemical composition of the methanolic extract of A. nilotica and indicates pyrogallol as the major polyphenol present in addition to the polysaccharide, polyphenol, amino acid, steroids, fatty acid esters, and triterpenoids. The in vivo antidiarrheal and ex vivo antispasmodic assays conducted in rodents indicate that A. nilotica possesses dose-mediated protection in mice from castor-oil induced diarrhea similar to loperamide while its preincubation in isolated rat ileum potentiated the isoprenaline-mediated inhibitory curves whereas the Ca ++ CRCs were shifted towards right with suppression of the maximum response, thus confirming its antispasmodic effect possibly mediated by a combination of PDE-inhibition and Ca ++ channels antagonist-like mechanisms, though additional mechanism(s) cannot be ignored.