ANTI-DIARRHEAL ACTIVITY OF ETHANOL AND CHLOROFORM SEED EXTRACT OF COLA NITIDA IN EXPERIMENTALLY INDUCED DIARRHEA

Objective: Cola nitida has been used in traditional medicine to treat diverse ailments including diarrhea. This study is carried out to investigate the antidiarrheal activity of the ethanol and chloroform seed extract of Cola nitida in Wistar albino rats. Methods: The ethanol and chloroform extracts of Cola nitida were evaluated with different doses (100mg/kg, 300mg/kg and 650mg/kg of animal weight) orally for antidiarrheal activity using castor oil induced-diarrhea, gastrointestinal motility test and castor oil-induced gastroenteropooling in Wistar albino rats. The observed activity was compared to standard antidiarrheal drug Lopermaide hydrochloride (2mg/kg) and to distilled water (10ml/kg) which served as the negative control. Results: Cola nitida ethanol extract at 150, 300 and 650mg/kg showed 55.64%, 59.73%, and 71.34% inhibition in gastrointestinal motility respectively. A significant reduction in diarrheal episodes (p<0.0001) was also observed with 650mg/kg of both extracts showing 100% inhibition. A reduction in the volume of fluid in the small intestine was also seen, this was however not significant. The chloroform extract of Cola nitida on the other hand produced a significant reduction in volume and weight of small intestinal content (p<0.05) with 650mg/kg showing a 92.73% inhibition of intestinal fluid accumulation. Conclusion: The ethanol and chloroform extract of Cola nitida showed anti-diarrheal activity in animal model by decreasing the frequency of defecation and by reducing gastrointestinal motility and intraluminal fluid accumulation in the intestine.


INTRODUCTION
Diarrheal disease is the second main cause of mortality in children below five years old, and is accountable for the mortality of approximately 525 000 children each year 1 . As a result of this, The World Health Organization set in motion a control program in 1988 to investigate traditional medical practices and other associated areas 2 . Diarrhea can continue for many days, and can cause the body to lose water and salts needed to survive. Formerly, the principal causes of deaths from diarrhea were severe dehydration and fluid loss. Certain factors including bacterial infections are now likely to account for an increasing number of all diarrhea-related deaths. Diarrhea occurring in undernourished children and people living with HIV could be potentially fatal 3 . In Ghana, it is the third main cause of mortality in children under the age of five 4 . Diarrhea has undesirable effects on the growth and development of cognitive ability in children 5 . An approximated 94% of the burden of diarrheal disease is attributed to the environment, and is associated with risk factors such as contaminated drinking water, low socio economic condition, lack of adequate sanitation and poor hygiene 6 . Cola nitida (Vent.) Schott and Endl. fruits have been employed traditionally as an aphrodisiac, appetite suppressant, to alleviate morning sickness, migraine, and indigestion 7 . It has also been used to relieve inflamed or wounded skin 8  most prevalent of the edible species 10 . The fruits are commonly used by students, drivers, and other menial workers to prevent hunger and thirst and as stimulant to keep awake and combat exhaustion 11 . Cola trees are best known for their seeds or nuts which are rich in caffeine and other secondary metabolites such tannins, phenols and xanthine 12 . It was also reported that the plant had antidepressant and antidiarrheal activity 11 . It is known that castor oil induces diarrhea through its active metabolite, ricinoleic acid which causes small intestine peristalsis which in turn leads to changes in the permeability of electrolytes in the intestinal mucosa 13 . Ibeh et al., evaluated the antispasmolytic and anti-diarrheal activity of the methanol extract of Cola nitida 14 and since cola nut is believed to possess antidiarrheal activity by traditionalists, this research is carried out to investigate the effect of the ethanol and chloroform crude extract of Cola nitida on diarrhea. When confirmed pharmacologically, Cola nitida stands the chance of further studies to isolate the active constituent responsible for activity.

MATERIALS AND METHODS Plant Material
Cola nitida seeds were obtained from Akwatia, a town in the Eastern Region of Ghana. The samples were identified and authenticated by Madam Anna Naa Quarley Quartey of Department of Pharmacognosy and Medicinal Chemistry School of Pharmacy, Central University, Ghana. A specimen of the sample was submitted to the University's herbarium with number CN003.

Plant Preparation and Extraction
The Cola nitida seeds were crushed into granules using mortar and pestle. A quantity of the comminuted granules equivalent to 2580.16g was extracted with ethanol (70%) and 1500g extracted with 1500ml of chloroform by cold maceration 15 . The mixture was shaken vigorously to enhance the extraction process and filtered after seven days to obtain the filtrate. The filtrate was evaporated using rotary evaporator (Drawell RE100 pro) to obtain the dry crude extract which was then stored in a refrigerator at 4 0 C until ready for animal experimentation.

Phytochemical Screening
The ethanol and chloroform crude extracts of Cola nitida were investigated for the presence of the following phytochemical constituents: phlobatannins, tannins, flavonoids, saponins, reducing sugars, alkaloids, cardiac glycosides using standard methods 15 .

Experimental Animal
Wistar albino rats (95-120g) of both sexes obtained from The University of Ghana Animal House were employed for this experiment. The rats were kept in standard plastic cages in a room with controlled 12hrs light and dark cycle. They had unrestricted access to clean water and were fed with standard pelleted commercial feed. The animals were allowed to acclimatize for 14 days before the experiments. The study was carried out according to the National Research Council Guide for the Care and Use of Laboratory Animals 16 and Organization for Economic Cooperation and Development (OECD) guidelines 17 .
The experiments were carried out in the Pharmacology Laboratory of Central University Ghana.

Acute Toxicity Study
The acute toxicity of Cola nitida was determined through the oral route. The rats were fasted for 24 hours and doses up to 2000mg/kg 17 of the ethanol and chloroform extract of Cola nitida were administered to rats of weight between the range of ninety to one hundred gram (90-100g) orally and rats were observed closely for the first six hours and subsequently periodically for seven days for mortality and any delayed toxic manifestations.

Gastro-Intestinal Motility Test
Gastrointestinal (GI) motility test was carried out according to standard methods 18 with slight modifications. Transit time of gastrointestinal content was measured at three doses of the ethanol and chloroform Cola nitida extract (150mg/kg, 300mg/kg and 650mg/kg) with distilled water (10ml/kg) as negative control and Loperamide hydrochloride (2 mg/kg) as positive control. All administrations were done orally with an oral gavage. All animals were administered 1ml of activated charcoal which served as a marker one hour after pretreatment. Animals were then sacrificed by cervical dislocation. The small intestines (from pylorus to caecum) were harvested and distance travelled by activated charcoal was measured and percentage inhibition of gastrointestinal motility was calculated.

Castor Oil-Induced Gastroenteropooling Test
The activity of Cola nitida on the inhibition of the accumulation of intraluminal fluid was ascertained by measuring the volume and weight of fluid accumulated in the small intestine over a period of time 21 . Rats were placed into five groups of five and pretreated as described above. One hour after pretreatment, rats were administered 1ml of castor oil and were sacrificed after another hour by cervical dislocation. The small intestine from the pylorus to caecum was harvested and the contents of each small intestine was emptied in a graduated measuring cylinder and weighed. The volume and weight was recorded and percentage inhibition of secretion was calculated.

Statistical Analysis
Statistical analysis was carried out using Graph Pad Prism 8.0. All data were summarized as mean±SEM (n=5). Multiple comparison tests were ascertained by one-way ANOVA along with post-hoc Tukey's honest significant difference (HSD) test. P< 0.05 was taken as statistically significant.

Phytochemical Screening
Results of different chemical tests on the ethanol and chloroform extracts of the seeds of Cola nitida showed the presence of flavonoids, tannins and other constituents.

Acute Toxicity Test
Administration of doses up to 2000mg/kg of the ethanol extract of Cola nitida orally did not produce any mortality nor any visible toxic manifestations. The chloroform extract of Cola nitida produced mortality at 2000mg/kg (50%), no deaths were observed when the dose was reduced to 1000mg/kg. Gastrointestinal motility test A significant dose-dependent inhibition of intestinal motility was observed by the ethanol (p<0.01 to p<0.001) and chloroform (p<0.05) extract of Cola nitida compared to the negative control as described in the table below. Loperamide hydrochloride produced the highest inhibition on gastrointestinal motility activity than the highest dose of both the ethanol and chloroform extracts.

Castor oil-induced diarrheal test
The ethanol (p<0.0001) and chloroform (p< 0.0001) extracts of Cola nitida significantly inhibited diarrhea with 650mg/kg of both extracts producing 100% inhibition of diarrhea. All doses of the ethanol and chloroform Cola nitida extracts significantly reduced the frequency of watery stools.

Castor oil-induced gastroenteropooling
The chloroform extract of Cola nitida caused a significant decrease in the volume (p=0.0039) and weight of small intestinal content (p<0.05) with 650mg/kg producing 92.73% reduction in volume of small intestinal content. A reduction was also observed with the ethanol extract, this was however not significant.

DISCUSSION
Plant or plant parts are used traditionally for the management of diarrheal episodes without any scientific evidence to validate their use. This study was conducted to assess the antidiarrheal activity of the ethanol and chloroform extracts of Cola nitida, which are considered to be effective in the management of diarrhea among the Ashantis in Ghana. Castor oilinduced diarrhea test model, gastrointestinal motility test and castor oil-induced enteropooling test were employed to ascertain the anti-diarrheal activity of Cola nitida extracts in this study. Castor oil-induced diarrhea test is employed to evaluate the anti-diarrheal effect of plants. Ricinoleic acid, the active constituent of castor oil is implicated in its diarrheal effect by stimulating peristaltic activity in the small intestine which leads to a change in permeability of electrolyte in the intestinal mucosa. It can also stimulate the release of endogenous prostaglandins which in turn result in the stimulation of secretion and motility 22 . Alkaloids, tannins, flavonoids and saponins are some of the phytochemical constituent present in the ethanol and chloroform extracts of Cola nitida (Table 1). Flavonoids have been reported to inhibit intestinal motility and prostaglandin synthesis by altering the synthesis of the cyclooxygenase enzymes 23, 24 . Tannins present in the ethanol and chloroform extracts of Cola nitida will form precipitates with the proteins present in the small intestine to form tannates which will in turn make the mucosa resistance to any chemical change and therefore reduce peristalsis and secretion 25,26 .  (Table 4). The chloroform extract was able to significantly inhibit the accumulation of intraluminal fluid relative to the control and the maximum reduction in volume of small intestinal content was shown by the highest dose of the extract. It can therefore be postulated that the inhibition of intestinal fluid accumulation observed may be as a result of the inhibition of the release of prostaglandin and consequently increasing the reabsorption of water and electrolytes. All doses of the ethanol extract of Cola nitida showed better anti-diarrheal activity in the gastrointestinal motility test and castor oil-induced diarrhea test than the chloroform extract which showed significant activity in the castor oil-induced gastroenteropooling test. This activity may be as a result of phytochemical constituents in the extracts working singly or together.

CONCLUSION
This study showed that the ethanol and chloroform seed extract of Cola nitida extract possessed significant anti-diarrheal activity which may be as a result of the presence of phytochemical constituents like tannins, flavonoids, saponins and alkaloids. This study therefore provides pharmacological basis for the use of Cola nitida for the management of diarrhea in some rural communities in Ghana.