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

Peace Doe*, Noah Kofi Ametepey¹, Vivien Chinenye Mshelia, Duke Bright Otchere, Omar Fofana, Audrey Antiwaa Amissah, Persis Nadine Yeboah, Harriet Naa Lamptey Mills, Ebenezer Ansah-Abrokwah¹

Department of Pharmaceutical Sciences, School of Pharmacy, Central University Ghana, P.O. Box 2305, Tema, Ghana.

DOI:  https://doi.org/10.22270/ujpr.v4i5.312

ABSTRACT

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. 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.

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. 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.

Keywords: Anti-diarrheal, Cola nitida, gastro-intestinal motility, seeds.

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 year¹. 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 areas². 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 fatal³.

In Ghana, it is the third main cause of mortality in children under the age of five⁴. Diarrhea has undesirable effects on the growth and development of cognitive ability in children⁵. 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 hygiene⁶. Cola nitida (Vent.) Schott and Endl. fruits have been employed traditionally as an aphrodisiac, appetite suppressant, to alleviate morning sickness, migraine, and indigestion⁷. It has also been used to relieve inflamed or wounded skin⁸. The bitter twigs of Cola nitida have also been used for teeth and gum cleaning⁹. Cola nitida is indigenous to West Africa and the nuts are obtained from cola trees. Cola has a broad number of species that have been widely cultivated some of which are Cola anomala, Cola verticillata (Thonn.) Stapf, Cola acuminata (Pal. de Beauv.) Schott and Endl. and Cola nitida (Vent.) Schott and Endl. are the most prevalent of the edible species¹⁰. 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 exhaustion¹¹.

Cola trees are best known for their seeds or nuts which are rich in caffeine and other secondary metabolites such tannins, phenols and xanthine¹². It was also reported that the plant had antidepressant and antidiarrheal activity¹¹. 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 mucosa¹³. Ibeh et al evaluated the antispasmolytic and anti-diarrheal activity of the methanol extract of Cola nitida¹⁴ 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 The 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 maceration¹⁵. 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 4⁰C 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 methods¹⁵.

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 Animals¹⁶ and Organization for Economic Cooperation and Development (OECD) guidelines¹⁷. 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/kg¹⁷ 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 methods¹⁸ 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 (2mg/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 Diarrhea Test

This test was performed according to standard methods ^19,20. Rats were fasted for 18 hours and were divided into five groups. The nature of fecal matter (put into three categories solid, semi-solid, liquid), and frequency of defecations were measured over a period of 6 hrs. Rats in the first group were administered distilled water (10ml/kg), group two received standard drug Loperamide hydrochloride (2mg/kg) while groups three, four and five received 100mg/kg, 300mg/kg and 650mg/kg of ethanol and chloroform extracts of Cola nitida seeds respectively. Castor oil (1 ml) was used to induce diarrhea in all experimental groups one hour after administration. Rats were placed in individual cages lined with absorbent paper. Percentage inhibition of diarrhea 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 time²¹. 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.

RESULTS

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 oil-induced 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 motility²². 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

This study showed that ethanol and chloroform extracts of Cola nitida had anti-diarrheal effect in all experimental models employed. In the gastrointestinal motility test, the extracts decreased the transit of charcoal meal dose dependently (Table 2). The ethanol extract at 150mg/kg, 300mg/kg and 650mg/kg showed higher inhibition of gastrointestinal motility (55.64%, 59.73% and 71.34%) compared to the chloroform extract at the same doses (5.13%, 10.48% and 12.02%). The percentage inhibition of gastrointestinal motility was comparable to that shown by the standard drug Loperamide hydrochloride. A reduction in motility in the gastrointestinal tract lengthens the time substances spend in the intestine thereby allowing for more water absorption²⁷. It can therefore be postulated that the reduction in gastrointestinal propulsion observed may be as a result of the anti-motility properties of the constituents present in the Cola nitida extracts. Studies have reported the anti-diarrheal activity of tannins and flavonoids as a result of their ability to reduce motility in the small intestine^28,23.

In the castor oil-induced diarrhea test, the ethanol and chloroform extract of Cola nitida produced significant decrease in the number of watery stools which may be due to its ability to inhibit the synthesis of prostaglandin stimulated by the action of castor oil. Maximum anti-diarrheal effect was seen with the highest dose (650mg/kg) of the ethanol extract of Cola nitida (Table 3) rather than the standard antidiarrheal drug Loperamide hydrochloride. This might also be as a result of the phytochemical constituents like tannins, alkaloids and saponins present in the extracts (Table 1) that may increase the time for water and electrolyte absorption by inhibiting intestinal motility²⁹.

For the castor oil-induced gastroenteropooling test, the chloroform extract of Cola nitida showed better activity compared to the ethanol extract (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.

ACKNOWLEDGEMENTS

The authors wish to appreciate the contributions of Mr. Kelvin for his help with the screening of phytochemical constituents and Mr. Kwame Koomson for his technical assistance all through the research.

Conflict of interest: No conflict of interest associated with this work.

CONFLICT OF INTEREST 

"No conflict of interest associated with this work”.

REFERENCES

1. WHO | Diarrhoea: why children are still dying and what can be done. WHO (2014).
2. Diarrhoeal Diseases Control Programme. (1988).
3. Riddle MS, Dupont HL, Connor BA. ACG clinical guideline : diagnosis, treatment, and prevention of acute diarrheal infections in adults. 2016; 111:602–622.
4. Osam–Tewiah EIC, Catherine EI. prevalence and management of diarrhoea in out- patient children less than 5years of age at the Princess Marie Louis Hospital (P.M.L.), Accra Ghana. (2010).
5. S. Department of Health and Human Services and Centers for Disease Control and Prevention. Diarrhea: Common Illness, Global Killer. Centers Dis. Control Prev. (2015).
6. Hons BP, Pharm D, Dip M, Pharm C, Psgh F. Children less than 5 years of age at the. 2010; 1–77.
7. Obey JK, Swamy TA. In vitro evaluation of antibacterial activity of infused Cola nitida Int J Curr Microbiol App Sci3, (2014).
8. Upton R. Herbal Medicines: A Guide for Health Professionals (Book Review). J Altern Complement Med 1998.
9. Esimone CO, Nworu CS, Adikwu MU, Odimegwu DC, Ezugwu CO. The effect of a new adaptogen, Garcinia kola seed, on the bioavailability of ofloxacin in humans. Sci. Res. Essay 2007; 2: 482–485.
10. Lovejoy PE. Kola in the History of West Africa. Cah. d’études Africaines 20, 97–134 (1980).
11. Tachie E, Brown N. Cola nitida and Cola acuminata. A state of Knowledge Report undertaken for the central African regional program for the environment 2006; 1-34.
12. Kanoma AI. et al. Qualitative and quantitative phytochemical screening of Cola Nuts (Cola Nitida and Cola Acuminata) 2014; 4(5):89-97.
13. Capasso F, Mascolo N, Izzo AA, Gaginella TS. Dissociation of castor oil-induced diarrhoea and intestinal mucosal injury in rat: effect of NG-nitro-L-arginine methyl ester. Br J Pharmacol 1994; 113: 1127.
14. Ibeh RC, Ikechukwu GC, Ijioma SN, Nwankwo CI, Singh AK, Asadi-Samani M, Onoja SO, Aloh GS. Anti-spasmolytic and anti-diarrheal activities of Newbouldia laevis, Cola nitida and Acanthus montanus extracts on gastrointestinal muscles. J Pharmacol Toxicol 2019;14: 1-8.
15. Trease and evans. Trease and Evans’ Pharmacognosy E-Book - William Charles Evans - Google Books. (2009).
16. Nih, Od, Oer, Guide laboratory animals for the care and use of eighth edition committee for the update of the guide for the care and use of laboratory animals institute for laboratory animal research division on earth and life studies 2011.
17. OECD/OCDE 423 OECD guideline for testing of chemicals acute oral toxicity-acute toxic class method introduction 2001.
18. Meite S et al. Anti-diarrheal activity of the ethyl acetate extract of morinda morindoides in rats. Trop J Pharm Res 2009; 8.
19. Awouters F, Niemegeers CJE, Lenaerts FM, Janssen PAJ. Delay of castor oil diarrhoea in rats: a new way to evaluate inhibitors of prostaglandin biosynthesis. J Pharm. Pharmacol 1978; 30, 41–45.
20. Uddin SJ et al. Antidiarrhoeal activity of the methanol extract of the barks of Xylocarpus moluccensis in castor oil- and magnesium sulphate-induced diarrhoea models in mice. J Ethnopharmacol 2005;101: 139–143.
21. Ezeja MI, Ezeigbo II, Madubuike KG, Udeh NE, Ukweni IA, Akomas SC,  Ifenkwe DC. Antidiarrheal activity of pterocarpus erinaceus methanol leaf extract in experimentally-induced diarrhea. Asian Pacific J Trop Med; 2012: 5(2), 147-50.
22. Pierce NF, Carpenter CC, Elliott HL, Greenough WB. Effects of prostaglandins, theophylline, and cholera exotoxin upon transmucosal water and electrolyte movement in the canine jejunum. Gastroenterology. 1971; 60, 22–32.
23. Carlo G, Autore A, Izzo AA, Maiolino N, Viola P, Diurno, MV, Capasso F. Inhibition of intestinal motility and secretion by flavonoids in mice and rats: Structure-activity relationships. J Pharm Pharmacol 1993; 45:1054–1059.
24. Hämäläinen M, Nieminen R, Asmawi MZ, Vuorela P, Vapaatalo H, Moilanen E. Effects of flavonoids on prostaglandin E 2 production and on COX-2 and mPGES-1 Expressions in Activated Macrophages. Planta Med 2011; 77: 1504–1511.
25. Tadesse E, Engidawork E, Nedi T, Mengistu G. Evaluation of the anti-diarrheal activity of the aqueous stem extract of Lantana camara Linn (Verbenaceae) in mice. BMC Complement. Altern Med 2017; 17: 190.
26. Kumar A, Upadhyaya K. Tannins are astringent. J Pharmacog Phytochem; 2012: 1.
27. Katzung BG, Trevor AJ. Basic and clinical pharmacology.12^th
28. Ojewole JAO, Awe EO, Chiwororo WDH. Antidiarrhoeal activity of Psidium guajava (Myrtaceae) leaf aqueous extract in rodents. J Smooth Muscle Res 2008; 44, 195–207.
29. Zia-Ul-Haq M. Antimalarial, antiemetic and antidiabetic potential of Grewia aslatica leaves. J Med Plants Res 2012; 6.

Table 1: Results of phytochemical screening of ethanol and chloroform seed extract of Cola nitida

Present (+); Absent (-) 

Table 2: Effect of ethanol and chloroform extracts of Cola nitida on inhibition of gastrointestinal motility

Mean±SEM (n=5). *P<0.05 **P<0.01, ***P<0.001, ****P<0.0001

Table 3: Effect of ethanol and chloroform extract of Cola nitida on castor oil-induced diarrhea in rats

Mean ± SEM (n=5). ****P<0.0001

Table 4: The effect of the ethanol and chloroform seeds extract ofCola nitida on castor oil-induced gastroenteropooling

Mean±SEM (n=5). *P<0.05, **P<0.01 compared to the control