Ethanol Extract of Spondias mombin L. Leaves Exhibits Antidiarrhoeal Activity by Stimulation of Na+–K+ ATPase, Inhibition of Prostaglandins or Suppression of Nitric Oxide

This study was to justify the acclaimed antidiarrhoeal activity of ethanol extract of Spondias mombin leaves (EESML) and to suggest probable mechanism of action. EESML was screened for its secondary metabolites. The diarrhoeal models involved randomized Wistar rats in 5 groups of 6 animals each. Animals in groups A&B (negative and positive control) were treated with normal saline and loperamide respectively while those in groups C, D and E received 100, 200 and 400 mg/kg body weight of EESML respectively. EESML contained saponins, alkaloids, avonoids, tannins, steroids, phenolics and glycosides. EESML lengthened the onset time of diarrhoea and as well caused reductions in the number, fresh weight and total number of wet feaces; and increase in the inhibition of defecations. EESML increased the intestinal activity of Na + –K + ATPase; the concentrations of intestinal Na + , K + , Cl - , total protein and glucose but decreased the concentration of nitric oxide of the diarrhoeal rats. The intestinal uid concentrations of Na + , K + , Cl - were dose dependently increased by EESML. EESML also increased the length of the small intestine. EESML possess antidiarrhoeal activity owing to the secondary metabolites, ability to enhance Na + – K + ATPase activity and electrolytes as well as suppression of nitric oxide.


Introduction
Diarrhoea, characterized by the frequent passage of liquid feaces, increased motility of the gastrointestinal tract, increased secretion and decreased absorption of uid, as well as loss of water and electrolytes, is one of the leading causes of death in the world. It comes behind pneumonia as the leading cause of death among children both of which accounts for an estimated 40 % child death across the globe annually [1]. Diarrhoea causes an estimated 5 million deaths in children less than 5 years of age per year equivalent to 1 childhood death per minute [1,2].
The continuous search of antidiarrhoeal principles from natural plant products from a number of medicinal herbs/plants that have been implicated in folklore medicine is therefore not a misplaced priority, and one of such invaluable plants is Spondias mombin. Spondias mombin L. (Anacardiaceae) is an evergreen tree distributed mostly in tropical areas like Nigeria, Ivory Coast, Cameroon, Brazil, Mexico and Peru. The plant is known as Hog plum in English. The native name in Nigeria is Ichikara in Igbo, Tsardarmasar in Hausa and Akika etikan or Iyeye in Yoruba [3]. Various parts of the plant including the leaves, fruits, stem bark, seed, pulp, root and ower have ethnomedicinal uses for the management or treatment of ailments [4]. The leaves and bark of S. mombin contain tannins, saponins, avonoids, sterols, quinones, and antioxidant chemicals [7] and there has been few claims on both in vitro and in vivo e cacy [5][6][7][8][9][10][11][12][13][14][15].
The antidiarrhoeal study on the plant is still scanty and without information on the probable mechanism of action. This study was therefore aimed to report the constituent secondary metabolites of S. mombin leaves and screen the extract on various models for its antidiarrhoeal activity and suggest the mechanism of action.

Plant Material
Plant material of the Spondias mombin voucher specimen (voucher number: UILH/001/1147) was obtained from Isaba Ekiti, Nigeria. The dried samples were then pulverized into ne powder with an electric blender (Trident Ltd, China]. Powdered plant material was stored in an airtight jar and further defatted with ethanol (25°C) and ltered using Whatman No. 1 lter paper. The ethanol solvent was recovered by evaporation using a rotary evaporator (Shanghai, China) that yielded a semisolid mass further lyophilized (New Brunswick) at 25°C for 24 hours. The obtained dry powder was reconstituted in physiological saline to give the doses of 100, 200 and 400 mg/kg body weight used in this study.

Animals
Wistar rats (130.7 ± 5.14 g) were obtained from the Animal Holding Unit of the Department of Biochemistry, University of Ilorin, Ilorin, Nigeria). The rats were kept in well-ventilated house conditions (Temperature: 28-30°C), photo-period (12-hr light, 12-hr dark; humidity: 45-55%). Animals were fed with pellets (Premier Feeds, Ibadan) and water ad libitum except when fasted. The animals were handled and provided care in accordance with the ethical guidelines on the Care and Use of Laboratory Animals as highlighted by the National Research Council, USA [22].

Acute Oral Toxicity Test
OECD method for acute oral toxicity was adopted. The general signs and symptoms of toxicity, intake of food and water and mortality were recorded for a period of two days and then for a period of 14 days [23].

Castor Oil-Induced Diarrhoea in Rats
The method described by Doherty [24] but with some modi cations was adopted in this model. Healthy Wistar rats fasted overnight for 18 hours were induced into diarrhoea following oral administering 1 mL of castor oil. Animals with diarrhoea after one hour of castor oil administration were completely randomized into ve groups of six animals each. Rats of group A (negative control) received 1 mL of normal saline orally while rats of group B (positive control) received 3 mg/kg of the standard drug, loperamide (orally). Animals in the groups C, D, and E (test groups) were orally administered equal volume of the extract corresponding to 100, 200 and 400 mg/kg body weight of the extract respectively. The animals were placed (individually) in metabolic cages on white clean preweighed Whatman lter paper, which was changed hourly. The severity of diarrhoea was assessed each hour for 6 hours during which the time of onset of diarrhoea, the total number of faeces, diarrhoeal faeces, total weight of faeces, and percentage inhibition of diarrhoeal defecation in each group were computed. The weight of the faeces was obtained from the difference in the preweighed Whatman lter paper and fresh weight of the stool.
The faeces dry weight was derived by drying out the fresh faeces in an oven (Uniscope Laboratory Oven, SM9053, Surgifriend Medicals, England) at 100º C till a constant weight was obtained. Water content of the faeces was also obtained as the difference in the fresh weight of the faeces and dry weight of the faeces. After the 6-hour exposure period, the animals were sacri ced to prepare small intestine supernatants.

Magnesium Sulfate-Induced Diarrhoea in Rats
The modi ed method described by Doherty [24] in Sect. 2.5 was adopted in this model except that magnesium Sulfate was used here.

Castor Oil-Induced Enteropooling in Rats
The procedure described by Robert et al. [25] was adopted for the castor oil-induced enteropooling study. Wistar rats fasted for 18 hours overnight were completely randomized into ve groups of six animals each (Table 3). Animals in all the groups were orally administered 1mL of castor oil. One hour later, Animals in Group A which served as the negative control received 1 mL of normal saline while those in groups B (positive control), C, D and E (test groups) were orally administered same volume corresponding to 3 mg/kg of loperamide (standard drug), 100, 200, and 400 mg/kg body weight of the extract respectively by oral treatment.
After 2 hours of these treatments, the rats were sacri ced according to the method described by Akanji and Yakubu [26].
The ends/edges of the pylorus and caecum of the small intestine were tied with thread. The small intestine was dissected, weighed and its content gouged into a measuring cup. The percentage of inhibition of the intestinal content was computed from the volumes and the masses. The differences between full and empty intestines were computed weighing the intestines again. The intestinal uid was also analyzed for Na + , K + and Cl − concentration. Values are mean of 6 replicates ± SEM, values with different superscripts across the rows for each parameter are signi cantly different at P < 0.05.

Magnesium Sulphate-Induced Enteropooling in Rats
The modi ed procedure described by Robert et al. [25] in Sect. 2.7 was replicated expect that magnesium sulphate was used in this case.

Gastrointestinal Motility Using Charcoal
The method described by Robert et al. [25] but with some modi cations was adopted. Wistar rats were fasted overnight for 18 hours prior to the commencement of the experiment. The rats were completely randomized into ve groups of six animals each. They were administered 1 mL of castor oil orally. One hour later, animals in the group A which served as the negative control received 1 mL of normal saline orally while those in group B were used as positive control and were orally administered 1 mL, corresponding to 5 mg/kg body weight of atropine sulphate. Animals in the groups C, D, and E were orally administered equal volume of the extract corresponding to 100, 200 and 400 mg/kg body weight. After 30 minutes of treatment, all the animals were again administered orally with 1 mL of charcoal meal (10% charcoal suspension in 5% agarose agar, prepared by thorough mixture of 10 g of charcoal powder and 5 g of agarose agar 100 mL distilled water). The animals were then sacri ced after 45 minutes of charcoal administration, using the diethyl ether as anesthesia as previously described by Akanji and Yakubu [26].

pH of Intestinal Fluid Secretion
The pH of the intestinal uid collected was read using a pH cooperative paper (strip), ranged 5-9. This strip is capable of different colour change within this pH range which can be compared to a standard colour for a given pH value as supplied with the kit by the manufacturer [32]. The pH paper was dipped into the sample and the colour that developed was checked against known standards [32].

Statistical analysis
Data were expressed as the mean of six determinations ± Standard Error of Mean (SEM). The normally distributed data were analysed using Duncan Multiple Range Test and One-Way Analysis of Variance was used to compare each test value with the control. The differences were considered statistically signi cant at p 0.05 (con dence level = 95%). All the analyses were done with SPSS version 20.0 software (SPSS Inc, Chicago, IL, USA).

Antidiarrhoeal Activity of the Ethanol Extract of S. mombin Leaves in Castor Oil-Induced Rats
The ethanol extract of S. mombin leaves signi cantly (P < 0.05) prolonged the onset time of diarrhoea. The total number of feaces as well as the number of wet feaces, fresh weight and water content of feaces were signi cantly (p < 0.05) decreased. There was a 67% and 80% inhibition of defecation recorded at 100 and 200 mg/kg body weight of the extract ( Table 2). The percentage inhibition of defecation (86%) as well as the fecal parameters of rats treated with 400 mg/kg body weight of the extract compared favourably with those of the rats administered 3 mg/kg body weight of loperamide hydrochloride ( Table 2). In addition, the extract signi cantly (P < 0.05) increased the intestinal activity of Na + -K + ATPase as well as the concentrations of intestinal Na + , K + , Cl − , total protein and glucose but signi cantly (P < 0.05) decreased the concentration of nitric oxide of the diarrhoeal rats in a dose manner when compared with the normal saline treated diarrhoeal rats ( Table 2). The 400 mg/kg body weight of the extract treated animals gave the profound activity when compared with the other treatment groups including that of the standard drug, loperamide ( Table 2).

Antidiarrhoeal Activity of the Ethanol Extract of S. mombin Leaves in Magnesium Sulphate-Induced Rats
Ethanol extract of S. mombin leaves signi cantly (P < 0.05) prolonged the onset time of diarrhoea and as well caused a signi cant (P < 0.05) reduction in number of diarrhoeal feaces by 64 %, 86 %, and 100 % at a dose of 100 mg/kg, 200 mg/kg, and 400 mg/kg body weight respectively (Table 3). Standard drug loperamide cause signi cant (P < 0.05) reduction in diarrhoeal feaces by 85% at a dose of 3 mg/kg body weight. Furthermore, the extract signi cantly (P < 0.05) increased the intestinal activity of Na + -K + ATPase as well as the concentrations of intestinal Na + , K + , Cl − , total protein and glucose but signi cantly (P < 0.05) decreased the concentration of nitric oxide of the diarrhoeal rats in a dose manner when compared with the normal saline treated diarrhoeal rats ( Table 3). The activity of the extract on these parameters was highest in the 400 mg/kg body weight of the extract treated animals when compared with the other treatment groups including that of the standard drug, loperamide (Table 3).

Antienteropooling Activity of the Ethanol Extract of S. mombin Leaves in Castor Oil-Induced Rats
The mass and volume of intestinal uid were signi cantly (P < 0.05) decreased whereas the inhibitions of intestinal uid accumulation signi cantly (P < 0.05) increased by all the doses of the ethanol extract of S. mombin leaves when compared with the normal saline treated rats ( Table 4). The actions of the extract were most profound with the 400 mg/kg body weight of the extract treated animals when compared with the loperamide treated animals. In addition, the extract resulted to a 50%, 54%, and 65% inhibitions of intestinal uid accumulation by the 100 mg/kg, 200 mg/kg, and 400 mg/kg body weight respectively while loperamide gave a 56% inhibitions when compared with the normal saline treated rats (Table 4).
Furthermore, the intestinal uid concentration of Na + , K + , and Cl − were equally increased dose dependently (Table 4).

Antienteropooling Activity of the Ethanol Extract of S. mombin Leaves in Magnesium Sulphate-Induced Rats
The ethanol extract of S. mombin leaves signi cantly (P < 0.05) decreased the mass and volume of the intestinal uid dose dependently when compared with the normal saline treated animals ( Table 5). The most profound reduction by the 400 mg/kg body weight that compared favourably well with the standard drug, loperamide. Moreover, inhibitions of intestinal uid accumulation signi cantly (P < 0.05) increased at 55%, 59%, and 62% by the 100 mg/kg, 200 mg/kg, and 400 mg/kg body weight respectively by the extract as well as 61% by loperamide when compared with the normal saline treated rats (Table 5).
Furthermore, the pH of the intestinal was decreased while the intestinal uid concentration of Na + , K + , and Cl − were increased dose dependently (Table 5). Values are mean of 6 replicates ± SEM, values with different superscripts across the rows for each parameter are signi cantly different at P < 0.05.

Antimotility Effect of the Ethanol Extract of S. mombin Leaves
The ethanol extract S. mombin leaves produced signi cant (P < 0.05) increase in the lengths of the small intestine by all the doses of the extract but the distance travelled by the charcoal meal was signi cantly (P < 0.05) decreased when compared to both the normal saline and atropine sulphate treated rat ( Table 6). The percentage inhibitions of peristalsis were 7%, 26% and 42% by the respective dose of 100, 200 and 400 mg/kg body weight of the extract. The 400 mg/kg body weight compared favourably with the 50% by reference drug treated rats (Table 6).

Discussion
The high mortality rate of Diarrhoeal has informed the continuous search of natural products from plants for antidiarrhoeal principles/compounds that can enhance increased mucosal absorption or decreased secretion and as well facilitate increase in resistance to ow via segmental contraction, decreased propulsion and peristalsis.
In the present study, dose-dependent inhibition of the rate of defecation as well as inhibition and prolonged/lenghtened onset time of diarrhoea, decreased faecal parameters (frequency of stool, total number of feaces, water content fresh weight, and number of wet feaces) suggest the profound antidiarrhoeal property of ethanol extract of S. mombin leaves. This might be by inhibiting prostaglandin that induces diarrhoea via enhanced contraction of the smooth muscle, vasodilation, and secretion of mucus in the small intestine [33,34]. This can be corroborated from the reversal in nitric oxide content known to have a protective effect (antiin ammation) on the gastrointestinal tract and preventing reabsorption [34,35]. This reversal suggests the antiin ammation as one of the probable mechanism with which the extract acted as an antidiarrhoeal agent. The marked increase in concentration of Na+, K+, Cland glucose by the extract suggest that the extract has protective effect on the intestinal mucosa against irritation and in ammation by castor oil by inhibiting release of prostaglandins to stabilize secretion of water and these electrolytes [36] as well as their motility or transport [37]. Furthermore, the reversal and resultant increase in the intestinal activity of Na + -K + ATPase by the ethanol extract of S. mombin leaves additionally substantiate the antidiarrhoeal activity of the plants. It is also possible that the mechanism the plant extract exhibited its antidiarrhoeal agent was by this action because it is known that castor oil, via ricinoleic acid compromise electrolytes transport and reduces absorption of Na + and K + as a consequence of either decreasing or inhibiting the activity of Na + -K + ATPase in the small intestine and colon [38]. Ethanol extract of S. mombin leaves could also have demonstrated its antidiarrhoeal activity probably by inhibition of prostanglandin synthesis and production of platelet activating factors by the extract.
Again, the dose-dependent inhibition of the defecation rate as well as inhibition and elongated onset time of diarrhoea, decreased faecal parameters (total number of feaces, water content, frequency of stool, fresh weight, and number of wet feaces) suggest the profound antidiarrhoeal property of ethanol extract of S. mombin leaves. The antidiarrhoeal property could be due to increased absorption of water and electrolytes as evidenced in the signi cant increase in concentration of Na + , K + and Cl − as well as the activity of Na + -K + ATPase. This further substantiate the probable mechanism of action of the antidiarrhoeal property of the extract by increasing the activity of Na + -K + ATPase through its de novo synthesis or might have an in uence on the NO/prostaglandin pathway [39]. This may be the reason that defecation and the activity of Na + -K + ATPase were relatively higher than the known reference drug, laporamide, one of the most e cacious and widely employed antidiarrhoeal drug that slows down or halt transit in the small intestine, reduce colonic rate of ow, and consequently increase colonic water absorption [40].
Reduction in both weight and volume of intestinal content by ethanol extract of S. mombin leaves suggests its antienteropooling activity in castor oil-induced experimental. These effects can be said to direct effects of inhibiting induced intestinal accumulation of uid by reducing electrolytes secretion and water in the small intestine. This suggest that the extract enhanced the reabsorption of electrolytes and water from intestinal lumen [41]. This can be corroborated by the increase in the Na + -K + ATPase activity in the present study by the extract and may as well describe the enhanced reabsorption of the electrolytes and water [42].
Furthermore, the dose dependent reduction in masses and volumes of intestinal uid in magnesium sulphate induced enteropooling in this study suggest that the ethanol extract of S. mombin leaves facilitated the reabsorption of electrolytes and water and consequently subdued the stimulated uid accumulated in the intestine. The reabsorption of electrolytes and water by the extract might probably be a resultant effect of the increased activity of Na + -K + ATPase in the present study [43].
Also in the present study, the suppression of the propulsive movement or gastrointestinal transit of charcoal meal suggest the capability of the extract in reducing the frequency of stools and further support the prolongation in the time required for the absorption of water and electrolytes in diarrhoeal conditions. This can be direct consequence of a decrease in peristaltic activity as evidenced by the peristaltic index computed, and ultimately reduction in the gastrointestinal motility. The inhibited gastrointestinal motility in the diarrhoeal condition by the extract of S. mombin leaves can be said to be through anticholinergic effect because anticholinergic agents are known to inhibit gastrointestinal hypermotility [41].
The antidiarrhoeal activity of ethanol extract of Spondias mombin leaves may therefore be attributed to the presence of some of these secondary metabolites especially avonoids, alkaloids, saponins and phenolics that are present in the extract. It is possible they might have acted singly or in combination through any of these mechanisms [41,42]. These provide a scienti c basis for the potential use of the extract in diarrhoeal conditions.
In conclusion, the overall result in this study substantiated the antidiarrhoeal activity of the ethanol extract of Spondias mombin leaves and further thus justi ed its traditional use in diarrhoea treatment.
The antidiarrhoeal activity might have been conferred by the avonoids, saponins, alkaloids and phenolics may have acted by stimulating the activity of the Na + -K + ATPase, inhibition of prostaglandins or suppression of nitric oxide.