Pharmacological Actions of Methanol Leaf Extract of Combretum paniculatum Vent. (Combretaceae) on the Gastrointestinal System

Department of Pharmacology & Toxicology, Faculty of Pharmaceutical Sciences, University of Nigeria, Nsukka 410001 Enugu, Nigeria. Department of Pharmacology & Toxicology, Faculty of Pharmaceutical Sciences, Enugu State University of Science and Technology, Enugu, Nigeria. Department of Pharmaceutical and Medicinal Chemistry, Faculty of Pharmaceutical Sciences, University of Nigeria, Nsukka 410001 Enugu, Nigeria.


Introduction
Diarrhoea is characterized with frequent passage of liquid faeces and it involves both an increase in the motility of the gastrointestinal tract, along with increased secretion and decreased absorption of fluid, and thus a loss of electrolytes (particularly sodium) and water. 1 It is one of the main causes of infant mortality in developing countries, 2 causing about 5 to 8 million deaths a year, mainly among children under five years of age. 3 The use of antimotility agents, antibiotics, electrolyte and fluid replacement therapies are currently the mainstay in acute diarrhea management. Gastric ulcer is a common ailment throughout the world, in which the gastric mucosa becomes damaged and perforations lead to bleeding which affects about 10% of the world population. 4 Some endogenous and exogenous factors, including acid, pepsin, stress, and noxious agents such as non-steroidal anti-inflammatory drugs (NSAIDs), Helicobacter pylori infection, smoking, and alcohol consumption are known to cause or aggravate gastric ulcer. 5 Despite advances, adequate remedy for the gastrointestinal disorders

Extraction of plant material
Pulverized plant material (2.5 kg) was extracted with 5 L of methanol by cold maceration for 48 h with intermittent agitation and thereafter filtered using sieve cloth and No. 1 Whatmann filter paper. The plant material was repeatedly washed with fresh solvent mixture until the filtrate became clear. The filtrate was transferred into a stainless tray and allowed to air-dry to obtain the methanol extract of C. paniculatum (MECP) as 164.2 g (8.21%), after which the extract was collected, weighed, transferred into an amber-coloured bottle and preserved in a refrigerator. The greenish coloured extract yielded 164.2 g (8.21%). The percentage yield of the extract was determined using the formula: Phytochemical screening of extract The powdered leaves was subjected to various phytochemical screening for the identification of constituents using standard methods. 11 Pharmacological tests Acute toxicity studies The acute toxicity test of the methanol extract of C. paniculatum (MECP) was determined in mice using the method of Lorke. 12 Briefly, the tests involved two phases. The first phase was the determination of the toxic range. The mice were placed in three groups (n = 3) and were given MECP (10, 100 and 1000 mg/kg; p.o.) solubilized in a solution of 3%, v/v Tween 80. The treated mice were observed for 24 h for the number of deaths. The death pattern in the first phase determined the doses used for the second phase. Since there were no deaths recorded in the first phase, a fresh batch of 4 mice received 1000, 1600, 2900, and 5000 mg/kg MECP orally. The treated animals were observed for lethality or signs of acute intoxication intermittently for 24 h. The LD50 is the geometric mean of the highest non-lethal dose and the least toxic dose.

Anti-diarrheal activity Gastrointestinal motility test
Twenty-five (25) Wistar rats were divided into five groups of five animals each and were fasted for 18 hours with free access to water. The animals were given 0.3 mL each of castor oil orally, one hour later, extract at 100 mg/kg, 200 mg/kg and 400 mg/kg body weight were administered orally to Groups 2, 3 and 4, respectively, Groups 1 and 5 received 2.5 mL/kg of 3% v/v Tween 80 and the standard drug, atropine, at 1 mg/kg per oral. After one hour, each animal was given 0.2 mL of freshly prepared charcoal meal via the oral route. One hour later, all the animals were sacrificed by chloroform anesthesia, the stomach dissected and the entire intestine excised. The total length of the intestine from the pylorus to the ileocaecal junction and the distance travelled by the charcoal meal were measured and the percentage of transit inhibition calculated. 13 The percentage transit inhibition was determined using the formula: Where T0 = Total length of intestine T1 = Charcoal distance of test group

Castor oil-induced enteropooling assay in rats
The assay was carried out following the method described by Robert. 14 Rats were fasted for 12 h but allowed free access to water. They were divided into the following five groups of five animals each. Group 1 received vehicle (2.5 mL/kg), Group 2 received loperamide (3 mg/kg), and Groups 3 to 5 received MECP (100, 200 and 400 mg/kg), respectively 1 h prior to the oral administration of castor oil (1 mL/rat). One hour later, the rats were euthanized with chloroform, the small intestines were removed after tying the pylorus and ileocaecal junction. The entire intestine with the content were weighed, the intestinal contents were collected into a graduated tube, and the volume determined.

Castor oil-induced diarrhea in rats
Twenty-five rats were fasted for 18 hours and divided into five groups of 5 animals each and labeled groups 1 to 5. The plant extract at doses of 100, 200 and 400 mg/kg body weight was administered orally to Groups 2, 3 and 4, respectively. Groups 1 and 5 received 2.5 mL/kg of 3% v/v Tween 80 and the standard drug, loperamide at 2.5 mg/kg orally, respectively. One hour after the pre-treatment, all animals received 2 mL of castor oil orally. The animals were kept in separate metabolic cages with a white paper beneath the cage to collect faeces. 15 The severity and consistency of diarrhea was monitored at 1, 2, 3 and 4 hours after the castor oil administration. The total number of diarrheic faeces and non-diarrheic faeces in the groups treated with the extract was recorded. The total diarrheic faeces for the control group were considered to be 100%. The results were expressed as a percentage of inhibition of diarrhea. 16 Inhibition

Indomethacin induced ulcer
The method as described by Urishidani,17 was used in this study. Indomethacin (50 mg/kg) was administered orally to all the animals 1 h after the various treatments. The animals were sacrificed by using chloroform 8 h after the indomethacin treatment.

Absolute ethanol induced ulcer
Animals were allotted to five groups and pre-treated as described above. One hour later, each animal was administered with absolute ethanol (5 mL/kg). The animals were sacrificed 1 h after the administration of absolute ethanol. 18

Measurement of ulcer index
In each of these study models, the stomach of the sacrificed rats was removed, opened along the greater curvature, and then rinsed under a stream of tap water. The stomach was pinned flat on a corkboard and was observed using a hand lens (×10 magnification). Erosions formed on the glandular portions of the stomach were counted and each given a severity rating on a 0-3 scale based on the diameter of ulcer (0, no ulceration; 1, ulcers ≤ 1 mm; 2, ulcers > 1 mm ≤ 2 mm; 3, ulcers > 3 mm). The total ulcer score for each stomach divided by a factor of 10 was calculated for each animal and expressed as ulcer index (U.I.) and the average taken as mean ulcer index for each group. [19][20][21] The degree of ulcer protection for each treatment group was calculated as a percentage for the mean ulcer index of the negative control group. 22 Acid neutralizing capacity The acid neutralizing capacity value for MECP (500, 1000, 1500 and 2000 mg/5 mL) was compared with the standard antacid Danacid [Aluminium hydroxide (250 mg) + Magnesium hydroxide (120 mg)/5 mL]. To the mixture, water was added to make up the total volume of 70 mL and then mixed for 1 min. Thereafter, 0.1N HCl (30 mL) was added into standard and test preparation and stirred for 15 min after which 2 drops of phenolphthalein was added and mixed. The excess HCl was immediately titrated with 0.5N sodium hydroxide solution dropwise until a pink colour was obtained. 23 The moles of acid neutralized was calculated by: Moles of acid neutralized = Acid neutralizing capacity (ANC) per gram of antacid = Statistical analysis Data obtained was analyzed by One-way ANOVA followed by Dunnett's multiple comparisons post-hoc test using GraphPad Prism version 7.0. The values are expressed as mean ± standard error of mean (SEM). P < 0.05 was considered statistically significant.

Results and Discussion
Phytochemical screening of the leaf of Combretum paniculatum revealed the presence of numerous constituents such as flavonoids, saponins, tannins, phytosterols, reducing sugars and phenolic compounds (Table 1). Antidiarrheal properties of medicinal plants have been found to be due to tannins, flavonoids, alkaloids, saponins, reducing sugar, sterol and terpenes. 24,25 Hence tannins, reducing sugars and sterols may have contributed to the observed antidiarrheal and antiulcer activities of MECP established in this study. Oral administration of the extract up to the dose of 5000 mg/kg caused no death of the mice and no severe signs and symptoms of overt toxic effect in the mice. This suggests that its median dose is above 5000 mg/kg. MECP is therefore, considered safe as postulated by Lorke (1983). 12 The MECP exhibited dose-dependent reduction in intestinal charcoal meal propulsion in rats. There was a 36.42% inhibition of intestinal motility at 400 mg/kg, while 100 and 200 mg/kg MECP produced 30.29% and 32.74% inhibitions, respectively. However, these inhibitions were not statistically significant. The standard drug, atropine, showed a significant inhibitory effect of 49.79% on intestinal motility as presented in Figure 1. Atropine is known to inhibit intestinal transit probably due to its anticholinergic effect 26 and drugs which inhibit intestinal motility can also possess anti-diarrheal activity. MECP inhibited intestinal motility dose-dependently, however, it was not significant, hence, antimotility effect may not be the major contributor to the antidiarrheal effect of the extract. Table 2 showed that MECP significantly (P < 0.01) reduced diarrhea induced by castor oil. The percentage inhibition of defecation of MECP at 400 mg/kg was 54.39% while loperamide caused 59.65% inhibition. There was also similar percentage inhibition of diarrheic dropping between MECP (80.00%) and loperamide (83.64%). Castor oil, or its active metabolite ricinoleic acid, which produces irritation and inflammation of the intestinal mucosa, thus leading to prostaglandins release, stimulates intestinal motility and diminishes Na + , Cl -, and water permeability in the intestine. 27 The MECP also showed a non-dose-dependent reduction in intestinal volume ( Figure  2). At 400 mg/kg, MECP produced the highest (65.90%) reduction, while 100 mg/kg exhibited the least reduction (48.28%) in the volume of intestinal contents, respectively. Values for the extract treated groups were significant relative to the control. This suggests that MECP may decrease water and electrolyte secretion to the intestinal lumen while promoting their absorption, which in turn could decrease intestinal overload and distension. In addition, this could lead to a decrease in intestinal motility (giving a longer time for absorption) and water contents of the fecal drops and hence overall reduction in the total number of defecation. This is consistent with the mechanism of action of loperamide for its antidiarrheal effect. 28 Non-steroidal anti-inflammatory drugs (NSAIDs) such as indomethacin have been reported to damage gastric mucosa by decreasing prostaglandin (PG) synthesis through inhibition of cyclooxygenase (COX) enzymes and COX-independent mechanisms, thereby inducing ulceration. 29 MECP showed a non-dose-related gastric mucosal protection against indomethacin-induced ulcer. Pretreatment of rats with MECP showed there was significant (P < 0.05) ulcer protection at 400 mg/kg (55.81%) and omeprazole (53.49%) when compared to negative control ( Figure 3) indicating that MECP could be valuable in the prophylaxis of gastric ulcer.  Oral administration of absolute ethanol in the animal model is destructive to the stomach tissue, since it penetrates rapidly and easily into the gastric mucosa, producing gastric lesions. 30 Such lesions are characterized by extensive submucosal edema, hemorrhage, desquamation of epithelial cells and infiltration of inflammatory cells, which are typical characteristics of alcohol injury in humans. 31 One hour after the administration of absolute ethanol to the different animal groups, the rats developed ulcer. MECP showed varying degree of ulcer protection but significance (P < 0.05) was seen at 200 mg/kg dose (68.52%) relative to the negative control ( Figure 4). Pretreatment with MECP decreased ulcer index and also increased Antacids have been effective in accelerating healing of duodenal and gastric ulcers. 32 The ulcer healing action of antacids is believed to be due to the neutralization of gastric luminal acid. 32 The extract produced a concentration-dependent increase in acid neutralizing capacity as compared with the standard. At a concentration of 2000 mg/mL, the MECP exhibited a significant increase in acid neutralizing capacity similar to the standard, danacid as presented in Table 3. This suggests that the acid neutralizing action may contribute to its gastroprotective effect.  Values are mean ± SEM., n = 5, *,**,*** Significant difference from negative control (P < 0.05, P < 0.01 and P < 0.001), respectively. MECP = methanol extract of C. paniculatum, NDF = mean number of diarrheic feces; NFe = mean number of feces.

Conclusion
The findings in this study, suggests that the methanol extract of C. paniculatum leaves has antidiarrheal and antiulcer activities, thus, justifying its traditional use in the treatment of diarrheal and ulcer. However, further studies are needed to identify the exact mechanisms and chemical compounds that are responsible for these pharmacological actions.