Antidiarrheal Effect of 80% Methanol Extract and Fractions of the Leaves of Ocimum lamiifolium in Swiss Albino Mice

Introduction Worldwide, in children of under five years of age, diarrhea is responsible for more than 760,000 annual deaths. It is treated with both modern drugs and traditional medicinal plants, including O. lamiifolium. But the use of this plant as an antidiarrheal agent is not scientifically validated. Therefore, this study was aimed to evaluate antidiarrheal efficacy of the plant. Method The leaf powder was macerated by 80% methanol and then fractionated using n-hexane, n-butanol, and distilled water. Antidiarrheal activity was evaluated through different models (castor oil-induced diarrhea, enteropooling, and motility) using onset, number of wet feces, fluid content, weight and volume of intestinal content, and motility as test parameters by administering treatment doses to groups of mice. Group I received 10 mL/kg of the dissolving vehicle, Group II received either loperamide or atropine, and Groups III-V received extract doses of 100, 200, and 400 mg/kg, respectively. One-way ANOVA was used to analyze the data, followed by Tukey's post-hoc test. Results The crude extract exhibited a significant effect on the fluid content of feces at all tested doses. Additionally, the n-butanol and distilled water fractions revealed significant effects on onset of diarrhea at 400 mg/kg (p < 0.05), while the n-hexane fraction showed significant effects on number of wet feces, onset, and fluid content of feces at all tried doses. The crude extract and all the fractions (at 200 and 400 mg/kg) decreased the weight and volume of intestinal content significantly. Similarly, both the crude extract and distilled water fraction at 400 mg/kg as well as n-butanol and n-hexane fractions at 200 and 400 mg/kg showed meaningful differences on peristaltic index as compared to the negative control. Conclusion The results revealed that the leaf extract of O. lamiifolium has an antidiarrheal activity, which supports the traditional medical practice.


Introduction
Diarrhea is a symptom characterized by the presence of watery or loose feces, occurring three or more times per day with increased frequency and decreased consistency [1]. It is the second leading cause of child morbidity and mortality in developing countries. Globally, it is estimated that there are 2.5 billion episodes and 760,000 diarrheal deaths annually in children under five years of age, accounting for 9% of the estimated total annual child deaths [1][2][3]. A review in 2021 estimated that the prevalence of diarrhea was 15.3% among children under five in sub-Saharan Africa [4]. In Ethiopia, diarrheal disease is a big issue, and it is one of the top 15 countries in which nearly three-quarters of child deaths occur due to diarrhea [1]. It is the country's second principal cause of death among children under five [5]. Although studies are reporting a decline in morbidity and death as a result of diarrhea, there are concerns about a potential rebound in the future due to ever-increasing urbanization [6].
Infectious agents (bacteria, viruses, and parasites) and noninfectious agents (irritable bowel syndrome, coeliac disease, and malignancies) may cause acute or chronic diarrhea. Acute diarrhea is the passage of loose stools more than three times per day for less than 14 days, while persistent diarrhea lasts between 14 and 30 days and chronic diarrhea persists for more than a month. Lack of hygiene, poverty, malnutrition, lack of good drinking water, and maternal education are the risk factors [2,7].
As indicated in ethnobotanical studies, O. lamiifolium is used as an antidiarrheal agent in Ethiopia [13][14][15][16]. But it may be ineffective, toxic, and may have drug interactions [35,36], which rationalizes this study for an efficacy test. Furthermore, the findings of this study may be used as the input for future research in this field. e dried leaves were crushed to a coarse powder.

Preparation of 80% Methanol Crude Extract.
Extraction was conducted based on the method used by Chalalai et al. [37]. e powdered leaves (1.2 kg) were mixed with 80% methanol in 1 : 5 a ratio. e powder was macerated for 72 h at room temperature with repeated shaking and then filtered using a muslin cloth followed by a filter paper (Whatman No. 1). e marc was re-extracted two times by adding a similar volume of fresh 80% methanol. e filtered fluid extracts were poured into a container and evaporated to dryness at 40°C and then lyophilized. en, the crude extract was stored in a refrigerator at 4°C until use.

Fractionation.
e methanolic crude extract was fractionated using distilled water, n-butanol, and n-hexane as fractionating solvents. e crude extract (100 g) was suspended in 350 mL of distilled water and was poured into a separatory funnel, and then the same volume of n-hexane was used to separate nonpolar phytochemicals. e mixture exhibited two layers, with the n-hexane layer being on top. e water part was collected in another beaker and fresh nhexane was added again. Overall, n-hexane-based extraction was carried out three times separately. All the n-hexane filtrates were collected in one beaker and allowed to be dried in an oven at 40°C. On the remnant (aqueous layer), another 350 mL of n-butanol was added and shaken. Again, the aqueous layer was at the bottom. e n-butanol filtrate was collected in a beaker. Another 350 mL of n-butanol was added two more times separately to get the n-butanol fraction. e final remaining residue was an aqueous fraction. All the n-butanol extracts were collected in one beaker and then dried in an oven at 40°C while the aqueous residue was dried in a lyophilizer. All dried extracts were kept in an airtight container in the refrigerator until use.

Experimental
Animals. An inbred either sex Swiss albino mice with an age range of 8-12 weeks and a weight range of 20-30 g were chosen (obtained from colonies in the animal unit of Department of Pharmacology, College of Medicine and Health Sciences, University of Gondar). ey were housed in plastic cages with wood chip bedding and allowed free to water and food. ey were kept in a room having light similar to the natural cycle (12 h on and 12 h off). Animals were allowed to adapt in the laboratory for 7 days before to the beginning of the experiment. Care and management of mice were performed according to OECD guideline 420 [38].

Animal Grouping and Dosing.
For each of the solvent extracts and for each model, mice (n � 30) were randomly assigned from Group I to Group V (six animals per group). Group I was assigned as a negative control (took 10 mL/kg distilled water for methanol, aqueous, and n-butanol extracts as well as 10 mL/kg 2% tween-80 for n-hexane extract). Mice of group II (positive control) were given loperamide 3 mg/kg for two diarrhea models (castor oil-induced and enteropooling) while atropine sulfate 1 mg/kg was given in the motility model. Animals in Groups III to V were given 100, 200, and 400 mg/kg doses of the crude extract, n-butanol, water, and n-hexane fractions.

Phytochemical Screening of Leaves of O. lamiifolium.
e presence or absence of flavonoids, anthraquinones, tannins, glycosides, saponins, terpenoids, phenols, alkaloids, and steroids was assessed for all extracts using standard evaluation methods [39].

Antidiarrheal Activity Determination
is model was conducted as described by Muluken et al [40]. For each extract, either sex of mice (n � 30) were randomly allocated to five groups and fasted food (pellet) but not water for 18 h. Dosing of extracts was performed as indicated in the grouping and dosing section. One h after dosing, castor oil (0.5 mL) was given orally, and then the mice were placed in a metabolic cage in which the floor was covered with a nonwetting transparent paper. e paper was changed every hour, and the mice were observed for four hours. During the experimental observation period (4 h), onset time of diarrheal stool, number of total stools, number of diarrheal stools, and weight of feces were recorded. Onset time was measured from the time of castor oil administration to the occurrence of the first diarrheal stool. Measurement parameters in the negative control group were taken as 100% to be used as a benchmark for the effect of extracts. Inhibition of diarrhea and defecation (percentage) were calculated using the following formula [41]: %Inhibition of Diarrhea � Mean number of wet stools of(control group − treated group) Mean number of wet stools of control group × 100, %Inhibition of defecation � Total number of feces in the (negative control − treated group) Total number of feces in the negative control × 100. (2)

Castor Oil-Induced
Enteropooling. e method used by Chitme et al. [42] was followed to observe the effect of the crude extract and fractions on intestinal fluid accumulation.
is effect was found through the measurement of weight and volume of small intestinal fluid.
irty mice were grouped into five groups (six mice per group) and then fasted for both food and water for 18 h prior to administration of the extracts. Mice were dosed as described in the grouping and dosing section. One hour after dosing the extract, 0.5 mL of castor oil was administered orally. After about an hour, each mouse was sacrificed using the cervical dislocation method, and the small intestine was cut after tying the cecum and pyloric ends. en, the weight of the intestine was measured. e volume of intestinal content was measured after expelling it into a graduated cylinder [42]. e empty intestine was reweighed again and the weight difference between the full and empty intestine was recorded. Percentage inhibition of weight and volume were calculated as follows: where MWIC-mean weight of intestinal content, MWICC-mean weight of intestinal content of the control group, and MWICT-mean weight of intestinal content of the test group. Moreover, where MVIC-mean volume of intestinal content, MVICC-mean volume of intestinal content of the control Evidence-Based Complementary and Alternative Medicine group, and MVICT-mean volume of intestinal content of the test group.

Gastrointestinal Motility Test Model.
Like in the abovementioned models, thirty mice were allocated to five groups and fasted food for 18 h. en, the doses were given as explained in the grouping and dosing part. One hour after dosing, castor oil (0.5 mL) was given orally to each mouse. After oral gavage of castor oil, the mice were observed for 1 h and then 1 mL of charcoal meal (5% charcoal suspension in 2% tween-80) was given orally. One hour later, the mice were sacrificed using the cervical dislocation method, and then the small intestine was removed carefully by opening the abdomen in a way that could not affect the charcoal meal travel. e intestine was laid straight on a clean table. e charcoal travel length from the pylorus to the cecum was determined using a measuring ruler and showed as a percentage of the length of the whole small intestine [43].
Peristalsis index(PI) � distance travelled by the charcoal meal total length of small intestine × 100, %of inhibitio � PI of negative control − PI of drug or extract treated PI of negative control × 100.
e in vivo antidiarrheal indices (ADI) were computed using the formula formed by an et al [44].
where D freq is the onset of diarrhea obtained from castor oil diarrhea model, calculated as follows: Dfreq � mean onset of diarrhea(in treated group − in the negative control group) mean onset of diarrhea in the negative control group × 100.
G meq is percent inhibition of motility, and P freq is percent inhibition of diarrhea.

Statistical
Analysis. SPSS (version 26) was used to analyze the data and the results were expressed as a mean-± standard error of the mean (SEM). ANOVA (one-way analysis of variance) and then Tukey's multiple comparison test were used to identify the presence or absence of differences between groups. Differences between groups were considered meaningful if the Pvalue was less than 0.05.

Yields of the Plant.
A total of 175 g of crude extract were obtained from the maceration of 1.2 kg of O. lamiifolium leaf powder (14.58% w/w). After fractionation of the crude extract by n-hexane, n-butanol, and distilled water, a total dried yield of 6.25 g (6.25% w/w), 20.5 g (20.5% w/w), and 73.25 g (73.25% w/w) were obtained, respectively. Table 1, many phytochemical constituents were identified in the crude extract. Of these, the n-hexane fraction contained many kinds of phytochemicals; whereas, the distilled water fraction contained only a few kinds of phytochemicals.

Castor Oil-Induced
Diarrhea. In this model, the crude extract showed significant effect on onset time of diarrhea, number of diarrheal feces, and weight of fresh feces at 400 mg/kg dose (p < 0.05). Furthermore, the crude extract showed significant effect on fluid content of feces in contrast to the negative control (p < 0.001).
e distilled water and n-butanol fractions showed significant effect on onset of diarrhea at 400 mg/kg dose compared with the negative control (p < 0.05). In comparison with the negative control group, the n-hexane fraction revealed significant effects on onset time of diarrhea, total number of feces, number of diarrheal feces, and fluid content of feces at all tested doses. Similarly, the fraction showed significant effect on the weight of fresh feces at 400 mg/kg dose (p < 0.001) ( Table 2).

Effects on Castor Oil-Induced Enteropooling in Mice.
At 200 and 400 mg/kg, the methanolic crude extract revealed a meaningful difference on the volume and weight of small intestinal content in contrast to the negative control. e distilled water, n-butanol, and nhexane fractions showed significant effects (at 200 and 400 mg/kg) on both volume and weight of small intestinal contents (Table 3). 4 Evidence-Based Complementary and Alternative Medicine

Effects on Castor Oil-Induced Intestinal Transit in Mice.
Both the crude extract and the distilled water fraction (400 mg/kg) showed significant effects on charcoal meal travel and peristaltic index parameters when correlated with the negative control. Similarly, the n-butanol fraction revealed significant effects on charcoal meal travel (400 mg/ kg) and on peristaltic index (at doses of 200 and 400 mg/kg). Meaningful differences were observed on administration of the n-hexane fraction for both charcoal meal travel and peristaltic index parameters at 200 and 400 mg/kg doses when correlated with the negative control (Table 4). Table (Table 5), the hexane fraction showed a greater ADI value compared to others. Based on ADI values, the crude extract and fractions were arranged in the increasing order as distilled water fraction < n-butanol fraction < crude extract < n-hexane fraction.

Discussion
e leaf of O. lamiifolium is customarily used for the treatment of diarrhea in Ethiopia [13][14][15][16]. However, antidiarrheal efficacy was not scientifically proven. So, the aim of this study was to validate the antidiarrheal efficacy of the leaf extract of O. lamiifolium using castor oil-induced diarrhea in mice.
Evidence-Based Complementary and Alternative Medicine castor oil, which is released upon the action of lipases in the small intestine, is a known agent to cause diarrhea [42,43]. Ricinoleic acid causes the release of prostaglandins through intestinal irritation and inflammation mechanisms, which in turn increases intestinal motility as well as the secretion of water and electrolytes. e other mechanism to cause these effects is by activating G protein-coupled prostanoid receptor (EP3) on intestinal smooth muscle cells [45]. Additionally, it inhibits sodium-potassium ATPase by forming sodium and potassium ricinoleate salts in the lumen which may result in an increase in intestinal permeability [46]. In the castor oil-induced diarrhea model, the crude extract produced a significant antidiarrheal effect on many measured parameters like the onset of diarrhea, number of wet feces, and weight of fresh feces at 400 mg/kg (p < 0.05), while all tested doses of the crude extract had a meaningful difference on fluid content of feces (p < 0.001). e distilled water and n-butanol fractions at 400 mg/kg dose produced significant differences only on onset of diarrhea, while nhexane fraction produced meaningful differences on all tested parameters. e high activity for n-hexane fraction in many parameters may be due to the presence of many secondary metabolites, including alkaloids, flavonoids, tannins, saponins, steroids, anthraquinones, and terpenoids. Anti-inflammatory agents like non-steroidal anti-inflammatory drugs (NSAIDs) prevent diarrhea through inhibition   [29]. erefore, antidiarrheal activity of the extract of the leaf might be through inhibition of prostaglandin synthesis. Furthermore, the presence of phytochemicals like terpenoids and steroids may account for antidiarrheal activity because these phytochemicals are known to inhibit prostaglandin E synthesis, reducing intestinal secretion [47,48]. erefore, the observed higher activity of the n-hexane fraction may be due to the availability of these phytochemicals in the fraction. e secretory component of diarrhea was assessed by the enteropooling model. e crude extract and all the fractions at doses of 200 and 400 mg/kg showed meaningful reductions in volume and weight of intestinal content as compared to the negative control. e increase in weight and volume of small intestinal content might be due to the activation of nitric oxide pathway through the effect of ricinoleic acid [49]. It is known that the presence of phytochemicals like flavonoids, terpenoids, alkaloids [50], and steroids [43] decreases the synthesis of nitric oxide. Tannins affect the activity cystic fibrosis transmembrane conductance regulator protein (a protein that transporters chloride ions from epithelial cells to the lumen) in a way that can reduce secretion in the small intestine and colon [51]. In addition, tannins reduce intestinal secretion by inhibiting intracellular Ca 2+ inward current [52]. Furthermore, phenols are known to have antioxidant activity, which may involve in inhibition of inflammation [53].
In the third antidiarrheal activity assessment method (gastrointestinal motility model), the crude extract and distilled water fraction at 400 mg/kg (p < 0.01) dose, as well as the n-butanol and n-hexane fractions at 200 and 400 mg/ kg doses revealed meaningful differences on peristaltic index as compared to the negative control. is antimotility activity may be due to the concerted effect of secondary metabolites. Flavonoids are known to inhibit intestinal motility [54]. Terpenoids also inhibit intestinal motility in the gut [55]. Studies revealed that tannins decrease peristaltic movements through inhibition of the intracellular Ca 2+ inward current [52].
Antidiarrheal index (ADI) is used to evaluate antidiarrheal effect using different parameters from different models. It is a combined figure of D freq (delay in onset), G meq, (reduction of gut meal motility as % inhibition), and P freq (a decrease in the number of diarrheal stools as % inhibition). For all the extracts, the value increases with the dose. e higher ADI value for the n-hexane fraction indicates its superior antidiarrheal effect as compared to other extracts. e activity difference between the fractions may be due to differences in the attraction of phytochemicals both in quality and quantity to the fractionating solvents. As indicated in Table 1, n-hexane fraction was able to localize many phytochemical constituents which may be a reason for its superior activity.

Conclusion
is study revealed the methanolic crude extract and solvent fractions of the leaf of O. lamiifolium have antidiarrheal activity. erefore, the findings of this study provide a scientific basis for the traditional use of the leaf of O. lamiifolium as an antidiarrheal agent in Ethiopian traditional medical practice.
Data Availability e datasets are available from the corresponding author upon reasonable request.
Evidence-Based Complementary and Alternative Medicine 7