Evaluation of In Vivo Antidiarrheal Activity of Solvent Fractions of Hagenia abyssinica (Rosaceae) in Swiss Albino Mice

Background Ethiopia has several medicinal plants that have been used for their antidiarrheal activity. Hagenia abyssinica is the most commonly used medicinal plant for the management of diarrhea in Ethiopia. Thus, this study's aim is to investigate the antidiarrheal effect of solvent fractions of H. abyssinica. Methods Antidiarrheal activity of extract fractions obtained from different solvents was evaluated by using small intestine transit, enteropooling, and castor oil-induced diarrhea animal models. In all animal models, the solvent fractions treated groups were treated with three different doses (100 mg/kg, 200 mg/kg, and 400 mg/kg) of the solvent fractions, while the negative control group was treated with a vehicle (distilled water), and positive control group was treated with loperamide. Results The acute toxicity test revealed that the LD50 of H. abyssinica is > 2000 mg/kg. In castor oil-induced, the solvent fractions of H. abyssinica (at 200 mg/kg and 400 mg/kg) significantly (P < 0.05–0.001) prolonged the stool frequency, reduced the weight of feces, and delayed diarrheal onset time as compared with the negative control group. The fractions produced a significant (P < 0.05) antimotility effect at the doses of 200 mg/kg and 400 mg/kg as compared to the negative control. All solvent fractions at the middle and higher doses showed a statistically significant dose-dependent reduction in the volume of intestinal contents and weight of the feces. However, the solvent fractions of H. abyssinica at a dose of 100 mg/kg failed to produce a statistically significant activity in all parameters (number of wet feces, the onset of diarrhea, and number of total feces) when compared with the negative control group. Conclusion The extract fractions obtained from different solvents have shown significant antidiarrheal activity. Thus, this finding supports the claimed traditional use of H. abyssinica leaves for the treatment of diarrhea.


Introduction
Diarrhea is a condition of increased intestinal emptying and increased water content in the stool. Generally, if defecation occurs more than 3 times a day, excretion of fecal with soft/ liquid consistency or a combination of both showed an abnormal condition in the defecation process [1]. Eighty percent of the Ethiopian population have depended on traditional medicine due to insufficient access to modern medicine, acceptability by the community, and low cost of herbal medicine [2].
Several medicinal plants that possess antidiarrheal activity are available throughout the world. e antidiarrheal effects of these herbs could be due to the attendance of phytoconstituents including flavonoids, alkaloids, saponins, terpenoids, steroids, and tannins [3]. Scientific investigation for the search on novel antidiarrheal compounds from medicinal plants like leaf extract of Osyris quadripartite [4],

Plant Materials.
e leaves of H. abyssinica were harvested in the Northwest part of Ethiopia, Amhara region of Kosoye, on February 12, 2019. en the collected leaves of H. abyssinica were washed using distilled water and dried at room temperature. e identification and authentication of the leaves of H. abyssinica were done by a botanist, and the voucher specimens (003ZDK/2019) were placed at the Department of Biology, University of Gondar, Ethiopia.

Extraction and Fractionation.
e leaves of H. abyssinica were washed with distilled water to remove dirt and dust, and the cleaned plant materials were dried at room temperature (25-27°C). e plant materials were grounded into a coarse powder with an electrical mill. erefore, the fine powder plant materials were macerated separately in methanol for roughly 72 hours, and then the plant materials were filtered using Whatman filter paper No. 1. Likewise, a fresh solvent was used to remacerate the marc, filtrates of each successive maceration were concentrated using a rotary evaporator. Lastly, the semidried residues were frozen in a refrigerator and dried using a lyophilizer (Labfreez, China) to entirely confiscate the remaining solvent [15,16]. Water, ethyl acetate, and chloroform solvents were used for fractionation of the crude extract of H. abyssinica. Distilled water was briefly added to the crude extract of H. abyssinica and dissolved by using a separating funnel. Chloroform was then added and shaken to dissolve the components. Similarly, on the aqueous layer, an equal volume of ethyl acetate was added to it. In both cases, two layers were separated. e subsequent chloroform and ethyl acetate layers were separated and exposed to evaporation by using a hot air oven (40°C). en, the dried solvent fractions of H. abyssinica were kept separately in a desiccator until being used for the experiment [17].

Preliminary Phytochemical Screening of Leaves Solvent
Fractions. Qualitative tests were done for the solvent fractions of H. abyssinica for the presence of phytoconstituents such as steroidal compounds, saponins, terpenes, flavonoids, tannins, alkaloids, and phenolic compounds, by procedures as described in Trease and Evans, 1989 [18].

Acute Oral Toxicity Test.
e acute oral toxicity of H. abyssinica was conducted according to the Organization for Economic Cooperation and Development (OECD) guideline No. 425 [19]. On the first day of the test, one female Swiss albino mice fasted for 4 hrs. en, 2000 mg/kg of the extract was administered by oral route using oral gavage. e mice were observed for the manifestation of behavioral and physical changes, and special attention was given during the first four hours. Depending on the results from the first mice, the next 4 female mice fasted for an estimated 4 hrs and then a single dose of 2000 mg/kg of the extract was given orally and followed in the same manner. e observation continued daily for a total of fourteen days [19].

Experimental Procedures and Designs.
Healthy Swiss albino mice of both sexes weighing 25-30 g were used for this experiment. e mice were retained in a plastic mice cage, with the provision of fed or feed with a standard diet and water. e mice were kept beneath a standard temperature, humidity, and 12 hours light and darkness cycle, and all groups of mice were acclimatized for about 2 weeks before the experiment [20]. roughout the study period, the mice were assigned into three groups, a positive control group, a negative control group, and a test group containing six mice per group. In all animal models, the first negative control groups received distilled water (10 ml/kg), the second positive control received loperamide (3 mg/kg), and the next three groups (II, III, and IV) received different doses (100 mg/kg, 200 mg/kg and 400 mg/kg) of H. abyssinica solvent fractions via the oral route. e doses of the solvent fractions of H. abyssinica were determined based on the result of the acute oral toxicity test. e middle doses of the solvent fractions of H. abyssinica were 1/10 of the limit dose, the higher doses of the solvent fractions were twice of the middle doses of the solvent fractions of H. abyssinica, and a half dose of the middle doses of H. abyssinica was the lower dose of the solvent fractions of H. abyssinica [19,21].

Castor Oil-Induced Diarrhea Model.
irty Swiss albino mice of either sex were deprived of food for 18 hours with free access to water and divided randomly into five groups, as mentioned above. After 1 hour of treatment with the vehicle (distilled water), solvent fractions (100 mg/kg, 200 mg/kg, and 400 mg/kg), and loperamide (3 mg/kg), diarrhea was induced by oral administration of 0.5 ml of castor oil to each mouse. Following their administration, the animals were placed individually into metabolic cages in which the floor was lined with transparent paper for the collection of fecal matter. e transparent paper was changed every hour for a total of 4 hours. e mice were then removed from their cages and the weight of feces was obtained by subtracting the weight of filter paper from the weight of feces and filter paper. e onset of diarrhea, the number of wet stools, the total number, and the total weight of fecal output were noted. Finally, the percentage of diarrheal inhibition, as well as the percentage of the weight of total fecal output, was calculated by using the following formulas [22]: percentage of diarrheal inhibition � mean number of wet stools (control group − treated group)x 100 mean number of wet stools of the control group . (1)

Castor Oil-Induced Intestinal Transit.
erefore, the experimental mice were fasted for 18 hours and had free access to water. e mice were divided into five groups and treated as described above. After 1 hour of treatment, 0.5 ml of castor oil was administered and 1 ml of 5% activated charcoal suspension in distilled water was administered orally. e mice were then sacrificed by cervical dislocation 2 hours after castor oil administration.
en, the small intestine was dissected out from pylorus to caecum and placed lengthwise on white paper. Finally, the distance traveled by the charcoal meal and the total length of the intestine was measured and expressed as a peristaltic index [23]. peristalysisindex (PI) � distance travelled by the charcoal meal total length of small intestine × 100, % of inhibition � PI of negative control − PI of drug or extract PI of negative control × 100. (2)

In Vivo Antidiarrheal Index (ADI).
e ADI for the solvent fractions of H. abyssinica was calculated by merging three parameters engaged from the abovementioned models. en, it was articulated based on the following formula [24]: Dfreq is the delay in defecation time as a percentage of negative control; Pfreq is the reduction in purging frequency in the number of wet stools as a percentage of the negative control; and Gmeq is the gut meal travel reduction as a percentage of negative control).

Castor Oil-Induced Enteropooling
Model. Swiss albino mice were deprived of food for 18 hours while water was allowed ad libitum. en, the mice were grouped and treated similarly as described above. One hour later, 0.5 ml of castor oil was administered to each animal. e mice were sacrificed by cervical dislocation 1 hour after the administration of castor oil; the small intestine of each animal was isolated and weighed. en, the intestinal content of all mice was collected by draining it into a graduated tube. After the removal of the intestinal content, the intestine of each mouse was reweighed. e volume of each intestinal content was measured [25]. en, percent reductions in weight and volume of intestinal content were calculated using the following formulas:

Statistical
Analysis. e data obtained from the experiments were expressed as mean ± standard error of means (SEM). Statistical analysis was done using statistical package for social sciences (SPSS) version 24. Between-and withingroup analyses were carried out by using one-way ANOVA, and subsequently Tukey's multiple comparison tests. Finally, the findings were considered significant when P value < 0.05.

e Percentage Yield of Plant Material Extraction.
At the end of the extraction, 153 (14.6%) grams of dried leaf extract were collected. e yields of the fractions were 47.8%, 29.8%, and 17.5% for the aqueous fraction, ethyl acetate fraction, and chloroform fraction, respectively.

Preliminary Phytochemical Screening of the Solvent
Fractions.
e phytochemical screening results obtained from the tests were presented in Table 1.

Acute Toxicity Test.
In acute toxicity study, leaves extract of H. abyssinica revealed no mortality at 2000 mg/kg body weight dose. After administration of the extract, the mice did not show any toxic effects like changes in behavioral activities such as anxiety, polyuria, diarrhea, seizures, and coma. us, the leaves extract of H. abyssinica at 2000 mg/kg body weight showed good safety and the LD 50 of the H. abyssinica extract is > 2000 mg/kg.

Effects of Solvent Fractions on Castor Oil-Induced
Diarrhea. As shown in Table 2, the chloroform solvent fraction and ethyl acetate solvent fraction exhibited a statistically significant reduction in the number of wet feces (P < 0.001 for both) and the number of total feces (P < 0.05, P < 0.01, respectively) at higher doses of the fractions (400 mg/kg) when compared with the negative control group. Similarly, 400 mg/kg doses of the three fractions significantly (P < 0.001) delaying the onset of defecation when compared with the negative control group. However, the chloroform fraction and ethyl acetate solvent fractions failed to exhibit a significant effect in all parameters at 100 mg/kg and 200 mg/kg doses as compared to the negative control. ough, the aqueous solvent fraction exhibited a significant effect in all parameters (in the number of wet feces, the onset of diarrhea, and the number of total feces) measured in this model at 200 mg/kg and 400 mg/kg doses when compared with the negative control group. e inhibitions of defecation (%) were 38.46%, 57.91%, and 78.00% by chloroform, ethyl acetate, and aqueous fractions, respectively, at 400 mg/kg of the solvent fractions (Table 2).

Effects on Castor Oil-Induced Intestinal Transit in Mice.
e middle and high doses of the solvent fractions of H. abyssinica (chloroform, ethyl acetate, and aqueous solvent fractions) significantly suppressed the gastrointestinal motility of charcoal when compared with the negative control group. Similarly, the small intestinal transit was significantly (P < 0.001) reduced by Loperamide 3 mg/kg with a percentage value of 67.10%. e reduction of gastrointestinal transit of charcoal (%) was 7.10%, 17.61%, and 31.70% for the chloroform fraction; 14.00%, 35.90%, and 51.92% for the ethyl acetate fraction; and 30.40%, 47.30%, and 58.83% for aqueous fraction at tested doses of 100 mg/ kg, 200 mg/kg, and 400 mg/kg, respectively (Table 3).

Effects on Castor Oil-Induced Enteropooling.
In the intestinal fluid accumulation test, the weight and volume of the intestinal contents were significantly reduced by the chloroform and aqueous solvent fractions at the tested doses of 200 mg/kg (P < 0.01) and 400 mg/kg (P < 0.001) when compared with the negative control group. e maximum percentage inhibition of the volume of gastrointestinal contents was detected at 400 mg/kg such as 40.70% (P < 0.001), 47.30% (P < 0.001), and 52.60% (P < 0.001) for aqueous solvent fraction, chloroform solvent fraction, and ethyl acetate solvent fraction, respectively. All fractions exhibited a statistically significant percentage reduction in weight of small intestine with the highest percentage reduction (40.70%, 53.00%, and 47.00%, respectively) at 400 mg/kg dose of the solvent fractions when compared with the negative control group (Table 4).

Antidiarrheal Index.
e determination of in vivo ADI revealed that the ADI increased with the dose for each fraction. Among the fractions, an aqueous fraction at its higher tested dose had the maximum ADI as compared to 200 mg/kg and 400 mg/kg doses of all fractions, but less than the ADI of Loperamide. e solvent fractions exhibited an ADI of 52.37, 72.17, and 74.14, respectively, for chloroform solvent fraction, ethyl acetate solvent fraction, and aqueous solvent fraction at a dose of 400 mg/kg, respectively, demonstrating a dose-dependent activity on the antidiarrheal index values (Table 5).

Discussion
Different medicinal plants with antidiarrheal activity have been studied by animal models (effect on gastrointestinal transit, electrolyte, and water secretion) [26,27]. e antidiarrheal activity of extract fractions obtained from different solvents of H. abyssinica has not been investigated. erefore, the current study was planned to evaluate the antidiarrheal activity of the solvent fractions of H. abyssinica via animal models such as castor oil-induced diarrheal model, antipropulsive, and antientropooling.
Castor oil-induced diarrhea is a commonly used method to evaluate the antidiarrheal activity of medicinal plants [28]. e ricinoleic acid that is released from castor oil through the lipase enzyme stimulates irritation in the gastrointestinal mucosa.
is irritation caused secretion of platelet-activating factor, nitric oxide, cyclic adenosine monophosphate, prostaglandin, and tachykinins, which are inflammatory mediators. e 4 Evidence-Based Complementary and Alternative Medicine inflammatory mediators stimulate intestinal motility and increase the secretions of water and some electrolyte. Several studies revealed that castor oil could induce diarrhea within one to two hours following administration of 0.1-0.3 milliliters of castor oil [29,30].
In the castor oil-induced diarrhea model, the higher doses (400 mg/kg) of all the solvent fractions exhibited a statistically significant activity in all parameters determined: the number of wet and total stools, the weight of wet stools, and the onset of diarrhea. e extract fractions obtained from different solvents could produce their antidiarrheal effect by antisecretory mechanism as it was apparent from the decrease in the total number of wet feces. Moreover, nonsteroidal anti-inflammatory drugs can inhibit castor oilinduced diarrhea apart from its inhibition of prostaglandin synthesis [31]. Likewise, the extract of H. abyssinica has also  138.50 ± 7.65a * * * 1.83 ± 0.33a * * * 3.60 ± 0.18a * * * 80.00 Data are expressed as mean ± SEM (n � 6); analysis was performed with One-Way ANOVA followed by Tukey test; a compared to negative control; b compared to loperamide 3 mg/kg; * P < 0.05, * * P < 0.01, and * * * P < 0.001; CF: chloroform fraction; EAF: ethyl acetate fraction; AF: aqueous fraction; negative controls received 10 ml/kg distilled water. Data are expressed as mean ± SEM (n � 6); analysis was performed with One-Way ANOVA followed by Tukey test; a compared to negative control; b compared to loperamide 3 mg/kg; * P < 0.05, * * P < 0.01, and * * * P < 0.001; CF: chloroform fraction; EAF: ethyl acetate fraction; AF: aqueous fraction; negative controls received 10 ml/kg distilled water.
Evidence-Based Complementary and Alternative Medicine revealed anti-inflammatory activities similar to nonsteroidal anti-inflammatory drugs [32]. erefore, it is reasonable to suppose that the antidiarrheal activity of H. abyssinica solvent fractions might be due to a reduction in the production of prostaglandin. e phytoconstituents like terpenoids have been reported to obstruct the synthesis of prostaglandin [33], which are identified to take part in the activation of gastrointestinal secretions [34]. As a result, the significant antidiarrheal activity observed by the extract fractions obtained from different solvents could be because of the occurrence of different phytoconstituents in the solvent fractions of H. abyssinica. e current finding is in line with previous similar studies [4,35,36]. e aqueous solvent fraction at its higher dose (400 mg/ kg) showed a maximum effect on the percentage inhibition of defecation (78.00%). e chloroform fraction, however, was found to be active either at the middle or at the higher dose. e insignificant activity of the solvent fractions at 100 mg/kg doses could be because of the incapability of the phytoconstituents to reach an adequate amount to elicit antidiarrheal activity. is argument is supported by the fact that activity would be apparent with an increasing dose of the extracts. e current finding is in agreement with previous studies in which the aqueous solvent fractions of numerous medicinal plants have reduced the number of stooling [4,37].
In the gastrointestinal motility model, the most effective and extensively used antidiarrheal drugs produced their effect by different mechanisms such as by reducing the intestinal motility and blocking the secretion of intestinal contents. e activated charcoal model is employed to investigate the activity of medicinal plants on gastrointestinal motility, and it serves as a marker [5]. e higher doses of the solvent fractions of H. abyssinica repressed the transit of charcoal meal or propulsive movement throughout the gastrointestinal tract that demonstrates the solvent fractions of H. abyssinica leaves could be able to decrease the frequency of stool. However, the lower doses of all tested doses of the solvent fractions and the middle doses of chloroform fraction and ethyl acetate fraction did not show a statistically significant decrement in the percentage of gastrointestinal motility, and this showed that the solvent fractions have less antimotility effect at the lower and the middle doses of the solvent fractions. Cholinergic activation causes diarrhea by Data are expressed as mean ± SEM (n � 6); analysis was performed with One-Way ANOVA followed by Tukey test; a compared to negative control; b compared to loperamide 3 mg/kg; * P < 0.05, * * P < 0.01, and * * * P < 0.001; CF, chloroform fraction; EAF: ethyl acetate fraction; AF: aqueous fraction; negative controls received 10 ml/kg distilled water.  [38]. is finding indicates that the solvent fractions have poor anticholinergic activity on gastrointestinal mucosa at the lower and the middle doses of the solvent fractions. e solvent fractions repressed the propulsion of activated charcoal, which showed the efficacy of the extracts in decreasing the vagal peristaltic movements of the gastrointestinal tract system.
ese also give details about the muscle relaxant activity of the solvent fractions. is pharmacological effect of the solvent fractions might be one of the more likely mechanisms for their antidiarrheal activities.
In the castor oil-induced enteropooling model, all the solvent fractions of H. abyssinica significantly decreased the intraluminal fluid accumulation as compared to the negative control group. e current finding agreed with previous similar studies [39,40]. e maximal activity of the solvent fractions was comparable with the standard drug loperamide, which is one of the most commonly used drugs for the treatment of diarrheal disorder [41]; as presented in the current study, loperamide successfully inhibited the induced diarrhea. e ricinoleic acid, which is the metabolite of castor oil, brings inflammation and irritation of the gastrointestinal mucosa, resulting in prostaglandins secretion. e secreted prostaglandins inhibit the reabsorption of water and NaCl 2 [42]. Accordingly, the solvent fractions significantly inhibit the gastrointestinal hypersecretion and enteropooling by decreasing gastrointestinal accumulation of fluid or via facilitating reabsorption of water and electrolytes. e antienteropooling effect of the solvent fractions may also be linked to the presence of secondary metabolites such as tannins, steroids, and flavonoids. e phytoconstituents like steroids and flavonoids block the secretion of prostaglandins; by this means, they block the release of prostaglandins and increase the absorption of some electrolytes. Tannins reduce the fluid secretion in the gastrointestinal by different mechanisms such as blocking cystic fibrosis transmembrane conductance regulator and calcium-activated chloride channel, by generating a protein-precipitating reaction to the intestinal mucosa and by free radical scavenging activity [33,43]. Indeed, it is well established that the liberation of ricinoleic acid from castor oil also causes irritation and inflammation of intestinal mucosa, leading to the release of prostaglandins E2, which results in stimulation of secretion. Similarly, the in vitro and in vivo experiments have shown that flavonoids, terpenoids, and saponins can decrease the gastrointestinal secretion stimulated by prostaglandins, thereby inhibiting secretion activated by castor oil [44].
H. abyssinica contains phytoconstituents such as tannins, phenols, flavonoids, saponins, glycosides, alkaloids, anthraquinones, and terpenoids, as tested by phytochemical screening tests. erefore, the possible antidiarrheal properties of the solvent fractions of H. abyssinica might be due to the abovementioned secondary metabolites.

Conclusion
e findings of the present study demonstrated that the solvent fractions of H. abyssinica possessed significant antidiarrheal activities. e antidiarrheal activities of the solvent fractions could probably be attributed to the presence of phytoconstituents in the H. abyssinica. us, this finding supports the claimed traditional use of H. abyssinica leaves for the management of diarrhea.
Data Availability e data sets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Ethical Approval
Ethical clearance was obtained from the research and ethics committee of the Department of Pharmacology, University of Gondar, with a Reference number (SOP 04-105-11). Experimental procedures were completed using Swiss albino mice according to the internationally accepted laboratory animal use and care guideline.

Conflicts of Interest
e authors declare that they have no conflicts of interest.