Citation: Adeniyi OS, Omale J, Omeje SC, Edino VO. Antidiarrheal activity of hexane extract of Citrus limon peel in an experimental animal model. J Integr Med. 2017; 15(2): 158–164.
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Diarrhea is defined as a disease condition in which a person passes loose or watery stool for three or more times during a 24-hour period. Statistics show that diarrhea kills about 2 195 children every day, a number greater than the deaths caused by malaria, acquired immune deficiency syndrome and measles combined. The frequency and severity of this disease are worsened by lack of access to clean water and hygienic disposal of human waste, malnutrition, poor hand-washing technique, dirty environmental condition and lack of access to affordable health care. The greatest proportion of deaths from diarrhea occur in Africa and South Asia. However, it is both preventable and treatable.
Citrus limon, commonly known as lemon, belongs to the family Rutaceae. Citrus fruits are mainly used by juice processing industries, where the peels are generally thrown away. However, several reports have shown that these peels have important medicinal benefits. Lemon peels contain pectin, which is capable of reducing plasma and liver cholesterol. Lemon peel has also been reported to be effective in curing kidney stone disease and can be used to prevent the disease and its recurrence. Literature shows that the hexane extract of lemon peel has antidiabetic activity. Furthermore, two of the active constituents of lemon peel, limonene and salvestrol Q40, possess anticancer properties.
Other research has shown that C. limon peel exhibited highly significant antimicrobial activity against Escherichia coli, Staphylococcus aureus, Proteus mirabilis, Klebsiella pneumonia and Pseudomonas aeruginosa. In developing countries, enterotoxins that are produced and secreted by bacterial organisms like Vibrio cholerae, Salmonella, Shigella and E. coli are the principal causes of diarrhea. Thus, the use of C. limon peel might prove effective in treating diarrhea. Diarrhea is also characterized by increased secretion of fluid into the intestine, reduced absorption of fluid from the intestine and alterations in intestinal motility, usually accompanied by increased propulsion. No studies were found that reported traditional medicinal uses of lemon peel in the treatment of diarrhea. Furthermore, there were no reports on the use of C. limon in the treatment of diarrhea. Therefore, with the aforementioned medicinal benefits of lemon peel, the authors of this research decided to investigate the effects of hexane extract of C. limon peel (HECLP) on castor oil-induced diarrhea in rats by evaluating the frequency of defecating wet feces, intestinal motility and gastric enteropooling.
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2 Materials and methods
Healthy male albino Wistar rats (100–150 g) were used. The animals were housed under standard controlled environmental conditions, with a 12-hour light/dark cycle. They were kept in wire meshed cages and fed with standard pellet diet (Vital Feeds, Grand Cereals LTD, Jos, Nigeria) and water ad libitum. The rats were allowed to acclimatize for two weeks before the experiments. This study was given ethical clearance by the Institutional Research and Ethics Committee of Kogi State University, Anyigba, Nigeria (KSU/CHS/REC/002/VOL2). Animals were handled as per the guidelines of the Committee, which is also in accordance with the internationally accepted principles for laboratory animal use.
Plastic experimental cages were prepared as follows: white blotting paper was used to line the floor of the cages and wire gauze was placed about 2 cm above the floor. Rats were placed in these cages 2 h daily for one week, to familiarize them with the experimental environment. For all the experiments, the animals were fasted for 18 h, but they had access to water.
2.2 Preparation of hexane extract
Fresh C. limon (lemon) was bought from Railway Market, Makurdi, Benue State, Nigeria. The peels were removed and cut into small pieces with a knife, dried completely under shade and ground with an electric grinder into coarse powder.
The coarse powder was soaked in hexane in a 500 mL flat bottom reagent bottle. It was kept at room temperature and allowed to stand for 10 d with occasional shaking and stirring. After this, the liquid hexane was filtered through cotton wool and then through filter paper. Then hexane was allowed to evaporate at temperature 40–50 oC yielding the extract of peel of C. limon.
2.3 Number of wet fecal pellets
In this experiment, oral administration of castor oil was used to induce diarrhea. A pretest for castor oil-induced diarrhea in rats was conducted and all rats responded by passing wet feces. Thus, the antidiarrheal activity of C. limon peel was evaluated according to the method previously described by Teke et al. The animals were fasted for 18 h and divided into 5 groups of 5 animals each. Each rat was put in a separate cage and given 1 mL castor oil. Unless otherwise noted, all treatments in this experiment were administered orally. After 30 min, animals in the different groups were treated as follows: group I received the vehicle alone (0.4 mL of 1% Tween 80); group II, loperamide (3 mg/kg); group III, HECLP (5 mg/kg); group IV, HELCP (10 mg/kg); group V, HECLP (20 mg/kg). The fecal pellets from each rat were counted every 1 h from the time of HECLP treatment for the first 4 h. The presence or absence of wet feces was noted for each animal.
2.4 Evaluation of receptor activities
After the first experiment, the animals recovered from the effect of intake of castor oil within one day (evidenced by passing out the normal solid feces after they were returned to normal diet). They were allowed to rest for 2 weeks, and then they were used for the second experiment. A dose of HECLP that produced the greatest significant reduction in wet fecal pellet output (20 mg/kg) was used as the working dose for this study. Four receptor blockers: prazosin (α adrenergic receptor blocker), propanolol (β adrenergic receptor blocker), nifedipine (Ca2+ channel blocker), and atropine (M3 receptor blocker) were used for this experiment. These four receptors have been reported to affect motility and secretions from the gut. We therefore aimed to investigate if HECLP acts (activate or inhibit) on any of these receptors to inhibit intestinal movement, fluid secretion and absorption. Therefore, if HECLP acts on any of these receptors to reduce the number of wet fecal dropping, then blocking it will abolish the antidiarrheal effect of HECLP. Twenty-five rats were divided into 5 groups of 5 rats each. Thirty minutes before induction with castor oil, each group was given a different pre-treatment: group I received the vehicle alone (0.4 mL of 1% Tween 80); group II, 10 mg/kg propranolol; group III, 1 mg/kg prazosin; group IV, 2.5 mg/kg nifedipine; and group V, 0.1 mg/kg atropine (intraperitoneal injection). After the pretreatment time, each animal was given 1 mL of castor oil and after another 30 min, each rat was given a dose of HECLP (20 mg/kg). Fecal pellet output was recorded every hour for 4 h.
2.5 Castor oil-induced enteropooling
The rats were allowed to recover and rested for another 2 weeks and then used for this experiment. They were fasted for 18 h and divided into 5 groups of 5 animals each. One hour prior to administration of castor oil (1 mL) each group was treated as follows: group I, received the vehicle alone (0.4 mL of 1% Tween 80) and served as the control group; group II received 5 mg/kg HECLP; group III received 10 mg/kg HECLP; group IV received 20 mg/kg HECLP; group IV served as standard and received 3 mg/kg loperamide. One hour after castor oil administration, the rats were sacrificed; the ends of the small intestine were tied with thread before it was removed and weighed. The intestinal content was collected into a graduated cylinder, where its volume was measured. The intestine was then reweighed and the difference between the full and empty small intestine was calculated.[14,15]
2.6 Intestinal motility
The method of Gamaliel and Akah was used to evaluate intestinal motility. A new batch of 30 rats were divided into 6 groups of 5 animals each. Each rat in group 1 was given 0.25 mL of 0.1% Tween 80. Groups 2, 3 and 4 received 0.3 mL of HELCP equivalent to 5, 10, and 20 mg/kg respectively. Group 5 received 5 mg/kg of standard drug atropine and group 6 received 0.3 mL castor oil. After 30 min, the rats were given 0.3 mL of freshly prepared charcoal meal (CM; 10% deactivated charcoal in 0.1% Tween 80). After another 30 min, the rats were sacrificed by cervical dislocation and the anterior abdominal wall was immediately cut open to dissect out the whole small intestine (pylorus to caecum). The length of the small intestine and the distance between the pylorus region and the front of the charcoal meal was measured. This distance was expressed as a percentage of the length of the small intestine.
2.7 Statistical analysis
Results are presented as mean ± standard error of mean. The Student t-test was used to test the significance of differences between two groups. The values were considered significant when P < 0.05. Data were analyzed using SPSS version 20.0 software (International Business Machines Corporation).
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3.1 Effects of HECLP on wet fecal pellet production
Results showed that administration of castor oil induced diarrhea in all the animals. All doses of HECLP produced a significant (P < 0.05) decrease in the total number of wet fecal pellets passed compared with the control group, in a dose-dependent manner. HECLP at 20 mg/kg yielded 34.2% inhibition, while the standard drug, loperamide, produced the greatest effect of 68.4% inhibition (Table 1).
3.2 Effects of receptor blockers on fecal pellet production
Results showed that pretreatment of animals with propanolol abolished the antidiarrheal activity of HECLP; propanolol caused a significantly higher (P < 0.05) wet fecal output despite being treated with HECLP. There was a reduction in the number of wet feces in rats treated with prazosin, but the difference was not significant (P > 0.05). Hence, HECLP treatment was still effective. Furthermore, the antidiarrheal effect of HECLP was observed in rats pretreated with nifedipine and atropine, thus, they did not interfere with the activity of HECLP (Table 2).
3.3 Effects of HECLP on enteropooling
Results showed that the treatments with 5, 10 and 20 mg/kg HECLP and the standard drug loperamide significantly (P < 0.01) reduced the volume of intestinal fluid relative to control animals treated with Tween 80. HECLP and loperamide also significantly reduced (P < 0.01) the weight of intestinal content. This reduction in weight is in line with the reduction in volume of intestinal content as shown in Table 3.
3.4 Effects of HECLP on gastrointestinal motility
Table 4 shows that the CM traveled an average of 34.2% ± 4.29% of the full length of the small intestine in rats treated with the vehicle (Tween 80) control. HECLP produced a significant (P < 0.05) reduction in the relative distance travelled by CM, in a dose-dependent manner. The highest dose of HECLP (20 mg/kg) reduced the movement to 21.40% ± 1.42% of the small intestine, which was a 36.33% reduction in transit distance compared to the control. The standard drug atropine produced a more significant (P < 0.01) reduction in intestinal movement compared to HECLP, while the castor oil treatment significantly increased (P < 0.05) the rate of intestinal movement relative to control as shown in Table 4.
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The castor oil-induced diarrhea model in experimental animals is commonly used because castor oil reliably causes an increase in the secretion of fluids and electrolytes in the intestinal lumen. Castor oil has a high concentration of ricinoliec acid, a product of recinoleate which is responsible for castor oil’s ability to induce diarrhea. Ricinoliec acid facilitates the release of endogenous prostaglandins in the intestine, which cause arachidonic-induced diarrhea, characterized by an increase in intestinal transit time and an increase in wet feces production.[18–20]
Diarrhea is a very common disease, especially in tropical countries, and causes millions of deaths in the world annually.[21,22] Diarrhea accounts for the death of 1 in 9 children globally, making diarrhea the second leading cause of death among children under the age of 5. The rate of death from diarrhea among children with human immunodeficiency virus (HIV) is 11 times higher than for children without HIV. The incidence of diarrhea is associated with poor maternal education, poor personal hygiene, and decreases with the age of the child. Diarrhea can last several days, seriously depleting the body of water and electrolytes that are necessary for survival. Most people who die from diarrhea actually die from severe dehydration and fluid loss. Therefore, the treatment of diarrhea is an important medical topic.
The present study revealed that HECLP possesses antidiarrheal effects at the doses used (5–20 mg/kg). Thus, lemon peel, which is often discarded, can have beneficial effects. Moreover, lemon is a fruit that is available everywhere in the world, therefore the use of lemon peel would provide a cheap, affordable and accessible means of treating diarrhea. A number of other plants have also been reported to possess antidiarrheal activities, such as Solanum paniculatum root, Pelargonium luridum (Andrews) sweet root, Vernonia amygdalina, Cymbopogon citrates leaves and Holarrhena antidysenterica seeds.[25–28]
In the enteropooling study, HECLP significantly reduced the volume of intestinal fluid accumulation, which was induced by castor oil. The inhibition of intraluminal fluid accumulation by HECLP might be due to inhibition of prostaglandin synthesis. The reduction in intestinal fluid content could also be due to an increase in water and electrolyte reabsorption through the intestinal mucosa. The ability of HECLP to inhibit intestinal fluid accumulation is similar to earlier reports on the antidiarrheal activity of the other plant materials, including root bark of Cordia africana and leaves of Maranta arundinacea Linn.[30,31]
The intestinal motility test using activated charcoal as a marker has been used for over 60 years as a simple and effective method of assessing the effects of laxatives on the gastrointestinal tract.[13,25,32] This experiment also revealed that HECLP reduced intestinal motility during castor oil-induced diarrhea. Thus it might possess the ability to relax intestinal muscle. Many drugs and medicinal herbal extracts that are used in the treatment of diarrhea possess the ability to reduce intestinal contraction and motility.[25,26,33,34]
Stimulation or inhibition of receptors on the intestine is responsible for the different activities observed, which include secretion and peristalsis. Stimulation of the α and β adrenergic receptors inhibits the rate and force of intestinal contraction.[35,36] This study reveals that when β receptor was blocked by propanolol, the antidiarrheal effect of HECLP was abolished. This suggests that HECLP might be exerting its antidiarrheal effect by stimulating this receptor. This is in line with earlier findings that showed that extracts of Indigifera pulchra, Rhizophora mangle and zinc sulphate exert their antidiarrheal effect by activating the β adrenergic receptor.[13,37,38] Furthermore, adrenergic pathway inhibits fluid secretion in the intestine.[39,40] Hence, stimulation of β adrenergic receptors might represent the pathway by which HECLP reduces intestinal motility and secretion during diarrhea.
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Previous reports have shown that C. limon peel possesses antimicrobial activities, which could be important in combating the pathogens, that are causative agents of diarrhea. Furthermore, this research had demonstrated that HECLP reduced the frequency of passage of wet feces, inhibited intestinal fluid accumulation and reduced intestinal motility by stimulating the β adrenergic receptors of the gut. We therefore suggest that C. limon peel extract should receive further attention as a potential effective, affordable and accessible treatment for diarrhea.
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The authors of this research wish to appreciate the support of the Technologists in Physiology Department Laboratory, for setting up the lab each time we needed to work. We also appreciate the technical advice of Dr. Emmanuel Titus Friday and the assistance of Silas, during hexane extraction of the lemon peel.
7 Competing interests
The authors declare that they have no competing interests.
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Table 1 Effects of HECLP on number of wet fecal pellet output in castor oil-induced rats Data are presented as mean ± standard error of mean, n = 5. * P < 0.05, ** P < 0.01, vs control group; △△ P < 0.001, vs loperamide group. HECLP: hexane extract of Citrus limon peel.
Table 2 Effects of blockers on fecal pellet output in castor oil-treated animals given 20 mg/kg hexane extract of Citrus limon Data are presented as mean ± standard error of mean, n = 5. * P < 0.05, ** P < 0.01, vs Tween 80 control.
Table 3 Effects of HECLP on castor oil-induced enteropooling Data are presented as mean ± standard error of mean, n =5. ** P< 0.01, vs Tween 80 control. HECLP: hexane extract of Citrus limon peel.
Table 4 Effects of HECLP on castor oil-induced small intestine transit Data are presented as mean ± standard error of mean, n = 5. * P < 0.05, ** P< 0.1, vs Tween 80 control. HECLP: hexane extract of Citrus limon peel; CM: charcoal meal.
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