Hindawi Evidence-Based Complementary and Alternative Medicine Volume 2018, Article ID 3037120, 10 pages https://doi.org/10.1155/2018/3037120

Research Article Antidiarrheal Activity of 80% Methanolic Leaf Extract of Justicia schimperiana

Belay Mekonnen ,1 Assefa Belay Asrie ,2 and Zewdu Birhanu Wubneh 2

1 Hospital Pharmacy, University of Gondar, Gondar, Ethiopia 2Department of , College of Medicine and Health Sciences, University of Gondar, Gondar, Ethiopia

Correspondence should be addressed to Assefa Belay Asrie; [email protected]

Received 20 July 2017; Revised 9 November 2017; Accepted 29 November 2017; Published 6 February 2018

AcademicEditor:RainerW.Bussmann

Copyright © 2018 Belay Mekonnen et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background. is one of the leading causes of preventable death in developing countries and mainly afects children and infants. It has been reported that the leaf of Justicia schimperiana is used as an antidiarrheal agent in Libo Kemekem district, northwest Ethiopia. Method. Te 80% methanolic leaf extract of J. schimperiana wasevaluatedforitsactivityagainstcastoroil- induced diarrhea, enteropooling, and gastrointestinal motility in mice. Results. Signifcant reduction (� < 0.001)inthetotal defecation and diarrheal drops was produced by all the test doses of the extract. Percentage inhibition of wet feces was 42.58, 65.07, and 74.96% at 100, 200, and 400 mg/kg doses of the extract, respectively. Te extract also signifcantly inhibited castor oil-induced enteropooling at all test doses. Te percent reduction in mean weight of intestinal contents was 66.96, 67.83, and 76.52% at 100, 200, and 400 mg/kg doses of the extract, respectively. Te extract signifcantly reduced gastrointestinal movement of charcoal meal as well at 200 (� < 0.01)and400mg/kg(� < 0.001)doses.Conclusion. In conclusion, the methanolic leaf extract of J. schimperiana hasanantidiarrhealactivityandthissupportstheuseofthisplantinthetreatmentofdiarrheainthetraditionalsettings.

1. Background of age die from diarrhea each year, of whom most are from developing countries. Of all child deaths from diarrhea, 78% Diarrheaisthepassageofthreeormoreloosestools.Itischar- occur in African and Southeast Asian regions [7]. acterized by increased gastrointestinal motility and secretion Many patients with a sudden onset of diarrhea have self- andadecreaseintheabsorptionoffuidandelectrolytes limited illness requiring no treatment. However, in severe [1, 2]. Based on the duration, diarrhea is classifed into 3 cases, dehydration and electrolyte imbalance are the principal types: acute diarrhea (duration < 2 weeks), persistent diarrhea risks, particularly in infants, children, and elderly patients, (duration from 2 to 4 weeks), and chronic diarrhea (duration thus requiring both nonpharmacologic treatments, such as of more than 4 weeks) [3]. Acute diarrhea is mainly caused oral rehydration therapy (ORT) and zinc supplements, and by enteric pathogens including viruses, bacteria, and parasites pharmacological treatments [8]. whereas most cases of chronic diarrhea result from functional Antimotility and antisecretory agents are the mainstay in or infammatory bowel disorders, malabsorption syndromes, the treatment of diarrhea. and their derivatives con- and [4, 5]. Pathogenic agents, such as Cryptosporidium, tinue to be widely used in the treatment of diarrhea. Diphe- Giardia lamblia, and enteropathogenic bacteria, are thought noxylate, , and are commonly used opi- to be the cause of persistent diarrhea [6]. oid antidiarrheals. Tere are also many other drugs that have Diarrheal disease is one of the leading causes of pre- antimotility or antisecretory efects on the intestine and can ventable death in developing countries, and it mainly afects be used for the treatment of diarrhea [9, 10]. children and infants [3]. According to the WHO and UNICEF agentscanreducetheseverityanddurationofinfectious reports, there are about 2.5 billion cases of diarrheal disease diarrhea [11]. Most of the enteropathogens which cause per- worldwide every year, and 1.9 million children below 5 years sistent diarrhea are treatable with antimicrobial therapy [12]. 2 Evidence-Based Complementary and Alternative Medicine

Generally, fuoroquinolones have become the drugs of choice diarrhea without scientifc proof for safety and efcacy. for the empirical treatment of acute diarrhea in adults. On Investigating the safety and efcacy of this plant in an animal the other hand, third-generation cephalosporins have been model could give valuable evidence of the claimed traditional considered the best drugs for the empirical treatment of use of the plant. Tus, the aim of this study was to evaluate the severe acute infectious diarrhea in children [13]. in vivo antidiarrheal activity of 80% methanolic leaf extract of Herbal medicines have been used for treating diarrheal J. schimperiana in mice. diseases, and it is estimated that up to 80% of the population in developing countries depend on traditional medicines for primary healthcare [14]. Tere are an enormous number 2. Methods of herbal medicines around the world that are claimed to be efective in treating diarrhea [8]. Amaranthus caudatus, 2.1. Chemicals and Reagents. Distilled water (Ethiopian Cofea arabica, Balanites rotundifolia, Boscia coriacea, Cis- Pharmaceutical Manufacturing, Ethiopia), loperamide HCl sampelos pareira, Plumbago zeylanica, Solanum hastifolium, (Bafna Pharmaceuticals, Ltd., India), castor oil (Shiba Phar- Berberis crataegina, Cornus mas, Ecballium elaterium, Mentha maceuticals and Chemicals, Yemen), methanol (Lab Chem- longifolia, Rhamnus cathartica,andTeucrium polium are icals, Avishkar, India), activated charcoal (Okhla Industrial some of those medicinal plants used in the treatment of Area,India),andatropinesulphateinj.(0.1%)(JeilPharm.Co. diarrhea traditionally in diferent societies [15–17]. Ltd., Korea) were used. Other reagents, such as glacial acetic Medicinal plants are a promising source of new antidiar- acid (Blulux Laboratories (P) Ltd., India), ammonia (Loba rheal drugs. For this reason, the WHO has encouraged stud- Chemie Pvt. Ltd., India), ferric chloride (Blulux Laboratories ies pertaining to the treatment and prevention of diarrheal (P) Ltd., India), Mayer’s reagent (Research-Lab Fine Chem diseases using traditional medical practices [18]. Currently Industries, India), hydrochloric acid (Blulux Laboratories (P) available drugs are linked with adverse efects and con- Ltd., India), chloroform (Nice Chemicals Pvt. Ltd., India), traindications [19, 20]. resistance is another challenge sulphuric acid (HiMedia Laboratories Pvt. Ltd., India), and to think about used in the treatment of diarrhea benzene (Blulux Laboratories (P) Ltd., India), were used in [21]. Te high incidence of diarrhea in developing countries the phytochemical screening test. coupled with limitations of currently available antidiarrheal drugs and poor healthcare coverage may make traditional 2.2. Collection of Plant Material. Te fresh leaves of J. medicines good alternative agents for the management of schimperiana were collected from a rural area around Gondar diarrhea. town called Blajig, northwest Ethiopia, in November 2015. Justicia schimperiana, also known by synonyms Adhatoda Meanwhile, the fresh aerial part of the plant was sent to and schimperiana and Gendarussa schimperiana, has diferent authenticated at the National Herbarium of Ethiopia, Depart- local names in Ethiopia including “dhumuugaa” in Afaan ment of Plant Biology and Biodiversity Management, College Oromoo, “Sensel” or “Simiza” in Amharic, and “Surpa,” “Ka- of Natural Sciences, Addis Ababa University, Addis Ababa, sha,” or “Keteso” in Sidama. It belongs to the family of Acan- Ethiopia. Te plant specimen (voucher number BM/001) has thaceae. Tis plant has several medicinal uses in diferent been deposited there in the National Herbarium. areas of Ethiopia. Ethnobotanical study reports showed that the plant is used in the treatment of various ailments such as evil eye, hepatitis B (jaundice), rabies, asthma, common 2.3. Preparation and Extraction of the Plant Material. Te cold, stomachache, diarrhea, tapeworm infestation, anthrax, leaves of J. schimperiana were cleaned with distilled water wound, external parasite, ascariasis, and skin irritation [15, and air-dried under shade at room temperature. Tere- 22–24]. Justicia schimperiana is used in the treatment of afer, the dried leaves were coarsely powdered using mortar malaria in Ethiopian folk medicine and experimental investi- and pestle. Ten, 800 gm of powdered leaves was weighed gations confrmed that the crude extract of the plant showed using an electronic balance and extracted by maceration an antimalarial activity [25]. Another experimental study also using 80% methanol for three consecutive days at room confrmed that chloroform, methanol, and aqueous fractions temperature with occasional stirring. Afer three days, the of the leaves of Justicia schimperiana had an antimalarial supernatant was fltered with Whatman flter paper No. 1. activity [26]. Justicia schimperiana has been also traditionally Te residue was remacerated and fltered in the same way used for the treatment of diabetes mellitus. One published twice. Te combined fltrates were concentrated in an oven ∘ experimental study determined that the aqueous leaf extract set at 40 C. Te dried extract was weighed and transferred of the plant produced a fall in glucose level in normal into vials and kept in a desiccator in the laboratory at anddiabeticmice[27].Furthermore,thephytochemical room temperature until the actual study procedures started analysis on the n-hexane extract of Justicia schimperiana [29]. showed the presence of alkaloids, polyphenols, favonoids, Normally,intraditionalmedicine,peopleusetheleavesof glycosides, saponins, triterpenes, and quinones [28]. J. schimperiana for the treatment of diarrhea afer smashing It has been reported that J. schimperiana is traditionally and mixing them with water as a juice, but here in this used for the treatment of diarrhea in Libo Kemekem district, studyweused80%methanolextractoftheplantbecause northwest Ethiopia. Te leaves of the plant are smashed and hydromethanolic solvents (especially 80% methanol) are mixed with water and then the juice is drunk for diarrhea usually better in extracting the most important chemical [23]. J. schimperiana hasbeenusedinthetreatmentof constituents of diferent plants [30, 31]. Evidence-Based Complementary and Alternative Medicine 3

2.4. Experimental Animals. Swiss albino mice, weighing 2.7. Acute Oral Toxicity Test. Acute oral toxicity test was con- 18–30 g, 6 to 8 weeks of age, were used. Te mice were bred ducted according to the OECD 425 guideline on fve female at the Animal House of the Department of Pharmacology, Swiss albino mice at a single oral limit dose of 2000 mg/kg University of Gondar, under standard conditions. Tey were body weight [35]. Fife female Swiss albino mice were ran- housed in plastic cages with sofwood shavings and chips as domly selected for the test. One animal was frst dosed beddings, in a room with 12 : 12 dark-to-light cycle, at room atthelimittestdoseandsurvivedinthe24-hourfollow- temperature and 55% humidity, and with free access to up. Ten, the other four additional animals were sequentially clean water and pelletized food ad libitum.Allmicewere dosed at 2000 mg/kg so that a total of fve animals were tested. acclimatized to the working (lab.) environment one week Te animals were observed individually for signs of toxicity prior to the experiment [32]. atleastonceinthefrst30minutesaferdosing,periodically during the frst 24 hours, and daily for an additional 13 days, 2.5. Grouping and Dosing of Animals. For each of the three for a total of 14 days. antidiarrheal activity test models, 30 mice were used. Te mice were randomly divided into fve groups of six mice each 2.8. Castor Oil-Induced Diarrhea in Mice. Tis test was done for each model. In all models, the negative control groups based on the method used by Awouters et al. [36]. Tirty mice were treated with the vehicle (distilled water, 10 ml/kg). Te were fasted for 18 hours and divided into fve groups with six positive controls were treated with loperamide 3 mg/kg (in animals in each group. Te frst group received distilled water castor oil-induced diarrhea and enteropooling models) and (10 ml/kg) and the second group received loperamide atropine 1 mg/kg (in gastrointestinal motility test model). (3 mg/kg), serving as negative and positive controls, respec- Te other groups (Groups 3, 4, and 5) in each model tively.Groups3,4,and5received100,200,and400mg/kg received 100, 200, and 400 mg/kg doses of the crude extract, of the extract, respectively. Afer one hour, all the animals respectively. received 0.5 ml/animal of castor oil orally. Te animals were kept in separate metabolic cages. Te severity of diarrhea 2.6. Preliminary Phytochemical Screening. Qualitative phy- was assessed for 4 hours. Te mean total number of feces tochemical screening tests were carried out based on the (dry and wet diarrheal droppings) was determined and methods used by Savithramma et al. and Jones and Kinghorn compared with the negative control group. Te total score [33, 34] to determine the presence or absence of alkaloids, of diarrheal feces for the negative control group was con- tannins, favonoids, saponins, phenols, terpenoids, steroids, sidered as 100%. Te percent inhibition of total defecation glycosides, and anthraquinones in the 80% methanolic leaf andthatofdiarrheawerecalculatedusingthefollowing extract of J. schimperiana. formulas:

% inhibition of defecation − = Total number of feces in the negative control Total number of feces in treated group × 100, Total number of feces in the negative control %inhibitionofdiarrhea (1) − = Total number of diarrheal feces in the negative control Total number of diarrheal feces in treated group Total number of diarrheal feces in the negative control × 100.

2.9. Castor Oil-Induced Enteropooling. Te method employed castor oil and the small intestine of each from the pylorus by Chitme et al. [37] was used to determine the efect to the caecum was isolated and weighed. Ten, the intestinal of the plant extract on castor oil-induced intestinal fuid content of all individual animals was collected by milking accumulation.Tirtymiceweredeprivedoffoodfor18hours into a graduated tube and the volume of each was measured. while water was allowed ad libitum.Ten,theanimalswere Just afer removal of the intestinal content, the intestine of grouped and treated in the same fashion as described for each mouse was reweighed. Ten, percent reductions in the castor oil-induced diarrhea above. One hour later, 0.5 ml weight of intestinal content and volume of intestinal content, of castor oil was administered per mouse in all groups. relative to the negative control group, were calculated using Te mice were sacrifced 1 hour afer the administration of the following formulas:

% reduction in weight of intestinal content ( ) − ( ) = Weight of intestinal content g in the negative control Weight of intestinal content g in treated group Weight of intestinal content (g) in the negative control × 100, 4 Evidence-Based Complementary and Alternative Medicine

% reduction in volume of intestinal content ( ) − ( ) = Volume of intestinal content ml in the negative control Volume of intestinal content ml in treated group Volume of intestinal content (ml) in the negative control × 100. (2)

2.10. Gastrointestinal Motility Test. Te mice were divided in all groups. Again, afer an hour, 0.5 ml of 5% charcoal into fve groups each containing six mice and fasted for 18 suspension in distilled water was given to each mouse orally. hours while water was provided ad libitum.Tefrstgroup Ten, all animals were sacrifced afer 30 minutes and the (control group) received distilled water orally (10 ml/kg), small intestine of each mouse was isolated. Te intestinal while the second group (positive control) received atropine length moved by the charcoal meal from the pylorus towards (1mg/kgIP).TemiceinGroups3,4,and5weregiven100, thecaecumwasmeasured,andthepercentofintestinallength 200, and 400 mg/kg of the plant extract, respectively. Afer travelled by the charcoal meal and percent inhibition of onehour,0.5mlofcastoroilwasgivenorallytoeachanimal intestinal transit were determined as follows [38]:

% inhibition of intestinal transit

% intestinal transit of charcoal meal in the negative control − % intestinal transit of charcoal meal in treated group = (3) % intestinal transit of charcoal meal in the negative control × 100.

2.11. In Vivo Antidiarrheal Index (ADI). In vivo antidiarrheal ADI �� V�V�=√3 � freq×�meq ×�freq, (4) index (ADI) of treated groups was determined using data from castor oil-induced diarrhea, enteropooling, and gas- trointestinal motility tests using the formula developed by where � freq is the delay in defecation time or diarrhea onset Aye-Tan as described below [39]: calculated as

mean onset of diarrhea in treated group − mean onset of diarrhea in the negative control group � freq = × 100, (5) mean onset of diarrhea in the negative control group

� meq is the charcoal meal travel reduction (as % of Committee, Department of Pharmacology, University of the negative control) from the gastrointestinal motility test Gondar, before the actual experimental activities were com- model, and � freq is the reduction in the number of wet stools menced. (as % of the negative control) from the castor oil-induced diarrhea model. 3. Results 2.12. Statistical Analysis. Data was analyzed using SPSS sta- 3.1. Preliminary Phytochemical Screening. Te preliminary tistical sofware, version 16. Results were expressed as means phytochemical screening test of 80% methanolic leaf extract ± SEM. Comparisons between groups were made using of J. schimperiana showedthepresenceofphenols,tannins, ANOVA followed by post hoc Tukey’s multiple comparison favonoids, saponins, terpenoids, glycosides, and anthra- test. At 95% confdence interval (� < 0.05), the diference quinones. between the compared groups was considered as statistically signifcant. 3.2. Acute Oral Toxicity Test. In the oral acute toxicity test using the limit dose of 2000 mg/kg body weight, the 80% 2.13. Ethical Consideration. Te experimental animals were methanolic leaf extract of J. schimperiana was found to be safe handled and cared for during the experimental procedures as there was no mortality or remarkable signs of toxicity noted according to the local ethical and internationally accepted in the 14-day observation of the mice which were used for the laboratory animal use, care, and welfare guidelines [32]. Te test. Tis result confrmed that the LD50 of the plant extract study was approved by the Experimental Animals Ethics is greater than 2000 mg/kg. Evidence-Based Complementary and Alternative Medicine 5

1.6 100 90 1.4 78.26 # 76.52

Ｎ 80 1.2 70 66.96 67.83 1 60 0.8 50 0.6 ∗ 40 ∗ 30 0.4 ∗∗ ∗∗ intestinal conten intestinal 20 0.2 % reduction in weight of in weight % reduction 10 0 0 0 Weight of intestinal content (g) content intestinal of Weight Group 1 Group 2 Group 3 Group 4 Group 5 Group 1Group 2Group 3Group 4Group 5 (a) (b) 1.2 100 80.65 1 80 76.34 # Ｎ 69.89 0.8 60 54.84 0.6 ∗∗ 40 0.4 ∗∗∗ ∗∗∗

∗∗∗ conten intestinal 0.2 20 % reduction in volume of of in volume % reduction 0

Volume of intestinal content (ml) content intestinal of Volume 0 0 Group 1 Group 2 Group 3 Group 4 Group 5 Group 1 Group 2 Group 3 Group 4 Group 5 (c) (d)

Figure 1: Efect of the methanolic leaf extract of J. schimperiana on castor oil-induced enteropooling in mice; (a) efect on the weight of intestinal content; (b) % reduction in the weight of intestinal content; (c) efect on the volume of intestinal content; (d) % reduction in ∗ ∗∗ ∗∗∗ thevolumeofintestinalcontent.Valuesexpressedasmean± SEM (�=6); � < 0.05; � < 0.01; � < 0.001 compared with the # negative control; % reduction is relative to the negative control group. Group 1: mice that received distilled water (negative control); Group 2: mice treated with 3 mg/kg of loperamide (positive control); Group 3: mice treated with 100 mg/kg of the extract; Group 4: mice treated with 200 mg/kg of the extract; Group 5: mice treated with 400 mg/kg of the extract.

3.3.EfectoftheExtractonCastorOil-InducedDiarrhea. A of intestinal contents. At 100, 200, and 400 mg/kg doses of signifcant reduction in the number of defecation instances the extract, the mean weights of the intestinal content were was observed with all the test doses of the extract compared 0.38 ± 0.11, 0.37 ± 0.07, and 0.27 ± 0.03 g, while the mean with the negative control group. Te mean total number of volumes of the intestinal content were 0.42 ± 0.11, 0.28 ± 0.08, defecation instances in extract-treated groups was 12 ± 0.93, and 0.22 ± 0.05 ml, respectively. Te highest dose of the plant 9.67 ± 0.88,and5.50 ± 0.76 at 100, 200, and 400 mg/kg doses extract (400 mg/kg) produced a more signifcant reduction of the extract, respectively, while this value was 4.33 ± 0.49 in in both the weight and the volume of the intestinal content. the loperamide-treated group. Te percent inhibition of total Te plant extract decreased the mean weight of the intestinal defecation relative to the negative control group was 38.99, contentby66.96,67.83,and76.52%andthevolumeofthe 50.84, and 72.04% at 100, 200, and 400 mg/kg doses of the intestinal content by 54.84, 69.89, and 76.34% at 100, 200, extract, respectively. Te standard drug had shown a more and 400 mg/kg doses, respectively, compared to the negative marked reduction in the number of defecation instances controls. Te highest percent inhibition, 80.65%, was scored (77.99%) compared to the vehicle-treated group. Te mean by loperamide (Figure 1). number of diarrheal drops in the extract-treated groups 3.83 ± 0.60 2.33 ± 0.42 1.67 ± 0.33 was , ,and at 100, 200, 3.5. Efect of the Extract on the Intestinal Transit of Charcoal and 400 mg/kg doses, respectively, while in the loperamide- Meal in Mice. Te plant extract reduced castor oil-induced 1.33 ± 0.21 treated group it was . Percent inhibition of wet gastrointestinal movement of charcoal meal signifcantly at (diarrheal) feces was 42.58, 65.07, and 74.96% at the doses of 200 (� < 0.01) and 400 mg/kg (� < 0.001)dosescompared 100, 200, and 400 mg/kg of the plant extract, respectively. Te with the negative control in a dose-dependent manner. Te percent inhibition of diarrhea recorded for the loperamide- highest efect was produced by 400 mg/kg dose of the extract, treated group was 80.06% (Table 1). which was comparable to the efect of the standard drug. Signifcant reduction in % intestinal transit of charcoal meal 3.4.EfectoftheExtractonCastorOil-InducedEnteropooling was produced by 200 (57.58 ± 6.65%, � < 0.05) and 400 mg/kg in Mice. Compared to the negative controls, the three serial (49.45 ± 2.54%, � < 0.01) and a more signifcant efect was doses of J. schimperiana leaf extract signifcantly inhibited produced by loperamide (40.10 ± 3.97%, � < 0.001), relative castor oil-induced enteropooling in mice at doses of 100, 200, tothenegativecontrolgroup.Comparedtothegroupthat and400mg/kgasrevealedbyreductioninweightandvolume received distilled water, the percent inhibition of intestinal 6 Evidence-Based Complementary and Alternative Medicine # - 65.07 74.96 42.58 80.06 diarrhea % inhibition of ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ feces 6.67 ± 0.88 1.67 ± 0.33 3.83 ± 0.60 2.33 ± 0.42 1.33 ± 0.21 Total number of diarrheal 0.23 ± 0.07 0.54 ± 0.12 0.32 ± 0.09 0.60 ± 0.11 0.24 ± 0.05 Weight of stool (g) # on castor oil-induced diarrhea in mice. - 77.99 72.04 38.99 50.84 defecation % inhibition of J. schimperiana inhibition is relative to the negative control group. Negative control: a group of mice that received distilled water. % # ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ 19.67 ± 0.88 4.33 ± 0.49 5.50 ± 0.76 Total number of feces ∗∗∗ ∗∗∗ compared with the negative control; (min) 66 ± 1.48 90.00 ± 7.64 9.67 ± 0.88 79.17 ± 2.30 12.00 ± 0.93 202 ± 6.25 130.00 ± 8.16 Table 1: Efect of methanolic leaf extract of Onset of diarrhea < 0.001 � ∗∗∗ ); 100 200 400 Dose �=6 (mg/kg) SEM ( ± Group Group 1 (negative control) - Group 2 (positive control) 3 Group 5 (extract) Group 4 (extract Group 3 (extract) Values expressed as mean Positive control: a group of mice treated with loperamide. Evidence-Based Complementary and Alternative Medicine 7

Table 2: In vivo antidiarrheal index of the methanolic leaf extract of J. schimperiana.

Dose Delay in diarrhea onset (� Charcoal meal travel Reduction in the number of In vivo Group (mg/kg) freq) reduction (� meq) wet stools (� freq) ADI Group 1 (negative ----- control) Group 2 (positive - 206.06 48.06 80.06 92.55 control) Group 3 (extract) 100 19.95 3.15 42.58 13.88 Group 4 (extract 200 36.36 25.42 65.07 39.18 Group 5 (extract) 400 96.97 35.95 74.96 63.93 Negative control: a group of mice that received distilled water. Positive control: a group of mice treated with loperamide (3 mg/kg) in a castor oil-induced diarrhea model and atropine (1 mg/kg) in a gastrointestinal motility test model.

45 100 35 ∗∗ 80 ∗∗∗ ∗ 25 ∗∗∗ 60 ∗∗ ∗∗∗ 15 40 charcoal meal charcoal

charcoal meal (cm) charcoal 20

5 of transit % intestinal Intestinal length travel by by travel length Intestinal 0 −5 Group 1 Group 2 Group 3 Group 4 Group 5 Group 1Group 2Group 3Group 4Group 5 (a) (b) 100 # Ｎ 90 80 70 60 50 48.06 40 35.95 30 25.42 20 10 3.15 % inhibition on intestinal transi intestinal on % inhibition 0 0 Group 1Group 2Group 3Group 4Group 5 (c)

Figure2:EfectofthemethanolicleafextractofJ. schimperiana on the intestinal transit of charcoal meal in mice; (a) efect on intestinal ∗ ∗∗ transit;(b)efecton%intestinaltransit;(c)%inhibitiononintestinaltransit.Valuesexpressedasmean± SEM (�=6); � < 0.05; � < 0.01; ∗∗∗ # � < 0.001 compared with the negative control; % inhibition is relative to the negative control group. Group 1: mice that received distilled water (negative control); Group 2: mice treated with 1 mg/kg of atropine (positive control); Group 3: mice treated with 100 mg/kg of the extract; Group 4: mice treated with 200 mg/kg of the extract; Group 5: mice treated with 400 mg/kg of the extract.

transit was 25.42 and 35.95% at 200 and 400 mg/kg doses of respectively, indicating a dose-dependent efect on the ADI the plant extract, respectively. Te highest percent inhibition value (Table 2). of intestinal transit of charcoal meal, 48.06%, was produced by atropine (Figure 2). 4. Discussion 3.6. In Vivo Antidiarrheal Index. Te antidiarrheal index Several studies have validated traditionally used antidiarrheal (ADI)isameasureofthecombinedvalueofdiferent plants by evaluating the efects of these plants on gastroin- parameters used to evaluate the antidiarrheal activity of a testinal transit and water and electrolyte secretion using given agent. Te extract showed an antidiarrheal index of animal models [40, 41]. Terefore, this study was proposed 13.88, 39.18, and 63.93 at doses of 100, 200, and 400 mg/kg, and conducted to evaluate the claimed antidiarrheal efect of 8 Evidence-Based Complementary and Alternative Medicine

Justicia schimperiana using antidiarrheal activity test models results can be taken as scientifc evidence showing that this in mice. plant has an antidiarrheal activity. Furthermore, the in vivo As shown in Table 1, the plant extract signifcantly (�< antidiarrheal index (ADI) of treated groups of mice, which 0.001) reduced the total fecal output and diarrheal drops in relates delay in onset of diarrhea, percent inhibition of wet castor oil-treated mice in the four-hour observation com- fecal output, and intestinal transit relative to the negative pared with the negative controls. Te three serial doses of the control group, indicates the dose-dependent efect of the extract were found to produce a reduction in the frequency plant extract [43]. But the exact mechanism of action of of defecation and diarrheal output to the same signifcant the plant extract for these efects was not determined. Te level that was produced by loperamide. Furthermore, at the possible mechanisms of action of the extract can be proposed highest dose, 400 mg/kg, the plant extract signifcantly (�< basedonthepathophysiologicprocessesofdiarrheaandthe 0.001) delayed the onset of diarrhea caused by castor oil when actions of castor oil to induce diarrhea. compared with the negative controls. Te percent reduction Diarrhea is caused by four pathophysiologic processes: in the frequency of defecation and diarrhea was increased increased luminal osmolarity, electrolytes secretion, de- with a corresponding increase in the dose of the plant extract. creased electrolytes absorption, and abnormal intestinal mo- Te percent inhibition produced by the highest dose of the tility causing reduction in intestinal transit time [44]. In extract was closer to the inhibition produced by loperamide. the intervention of diarrhea, antimotility and antisecretory Tis increasing pattern of percent inhibition in the total agents remain as the main agents used to decrease such number of fecal output instances and diarrheal episodes with pathophysiologic changes [45]. Castor oil has been widely increasing dose of the extract plus signifcant delay in the used for induction of diarrhea in antidiarrheal activity studies onsetofdiarrheabythehighestdoseimpliesthattheplant because it releases ricinoleic acid, a metabolite that causes extract inhibits diarrhea more efectively at relatively higher diarrhea, upon in the gut [46]. Ricinoleic acid doses [42]. initiates diarrhea via mechanisms such as irritation of GI Similartothefndingsinthecastoroil-induceddiarrhea mucosa,leadingtothereleaseofprostaglandinwhichstimu- model, all doses of the plant extract showed a signifcant lates gastrointestinal motility and electrolyte secretion, reduc- reduction in both the mean weight and the volume of intesti- ing electrolyte absorption from the intestine and colon; these nal fuid compared with the negative controls. Te percent are similar to the pathophysiologic processes resulting in reduction in weight and volume of intestinal content was diarrhea [47]. Terefore, the antidiarrheal activity of the plant increased with the dose of the extract. Tis result demon- might be due to the activities that oppose the actions of castor stratedthattheefectoftheplantextractonpercentinhibition oil for induction of diarrhea or pathophysiologic processes of castor oil-induced enteropooling is increased as its dose leading to diarrhea. Te extract has been shown to decrease was increased. Te results in this model revealed that the the intestinal fuid accumulation. Tis suggests that the plant efect of the highest dose of the extract on enteropooling extract may decrease water and electrolyte secretion to the (intestinal fuid accumulation) was found to be closer to and intestinal lumen while promoting their absorption, which in comparable with the inhibitory efect of loperamide. Te turn could decrease intestinal overload and distension, lead- fndings in this model may indicate that the plant extract ing to a decrease in intestinal motility (giving a longer time for has a signifcant antisecretory efect and this contributes to absorption) and water contents of the fecal drops and hence its antidiarrheal efect noted in castor oil-induced diarrhea overall reduction in the total number of defecation instances model. and diarrheal drops in treated groups. Tis is consistent In the gastrointestinal motility test model (Figure 2), the with the mechanism of action of loperamide for its antidiar- 80% methanolic leaf extract of J. schimperiana reduced gas- rheal efect as presented in the literatures [48]. In addition, trointestinal motility in castor oil-treated mice as shown by the extract may have an anticholinergic activity and cause reduction in gastrointestinal movement of charcoal meal. Te reduction in intestinal motility and secretion, which is in extract caused a signifcant reduction in the gastrointestinal agreement with the action of atropine on the intestine [49]. charcoal meal transit at 200 (� < 0.01) and 400 mg/kg (�< Te methanolic leaf extract of J. schimperiana contains 0.001) doses compared with the negative controls. Te efect phenols, tannins, favonoids, saponins, terpenoids, glyco- ofthesmallestdoseoftheextractonthisparameterwasfound sides, and anthraquinones as asserted by preliminary phyto- to be insignifcant. Te percent inhibition in intestinal transit chemical screening tests, and most of these secondary metab- was 3.15, 25.42, and 35.95% at 100, 200, and 400 mg/kg doses olites were reported to have an antidiarrheal activity. Reports of the extract, respectively. Tese fndings indicate that the in the literatures showed that tannins have an antispasmodic gastrointestinal motility was greatly decreased as the dose of and efect, favonoids inhibit prostaglandin the extract was increased. Te reduction in gastrointestinal E2-induced intestinal secretion, saponins inhibit histamine motility increases the time of stay of gastrointestinal contents release, terpenoids inhibit the release of prostaglandins, and in the intestine and this may promote intestinal water and phenols reduce intestinal secretion and transit and have an electrolyte absorption. So, the antidiarrheal efect of the plant astringent action. All these actions lead to the inhibition of extract may be attributed to, at least in part, its antimotility diarrhea by decreasing intestinal secretion and motility [50– activity. 52]. Terefore, the antidiarrheal activity of the plant extract In this study, the methanolic leaf extract of J. schimperi- may be produced by these chemical constituents. Moreover, ana has been shown to reduce castor oil-induced diarrheal the plant extract was found to be safe as no sign of toxicity episodes and intestinal secretion and motility. So, these was noted in the acute oral toxicity test. Tis indicates that the Evidence-Based Complementary and Alternative Medicine 9 plant is tolerable and safe even at higher doses than used in [4]W.C.L.Fischer,I.Rudan,L.Liuetal.,ChildhoodPneumonia the three antidiarrheal models of this study. Tis validates the and Diarrhea, Global Burden of Childhood Pneumonia and safety of the plant in its use in the traditional settings as well. Diarrhea Baltimore, USA, 2013, http://www.thelancet.com. [5] T. V. Nguyen, P. L. Van, C. L. Huy, K. N. Gia, and A. Weintraub, “Etiology and epidemiology of diarrhea in children in Hanoi, 5. Conclusion Vietnam,” International Journal of Infectious Diseases,vol.10,no. 4, pp. 298–308, 2006. In conclusion, the methanolic leaf extract of J. schimperiana has an antidiarrheal activity as revealed by reductions in the [6]A.K.Shrivastava,S.Kumar,N.K.Mohakud,M.Suar,andP.S. Sahu, “Multiple etiologies of infectious diarrhea and concurrent total fecal output and diarrheal drops, intestinal fuid accu- in a pediatric outpatient-based screening study in mulation, and gastrointestinal motility. Hence, this study Odisha, India,” BMC Gut Pathogens,vol.9,article16,2017. supports the use of the plant in the treatment of diarrhea in [7] WHO, Departments of Child and Adolescent Health and Devel- the traditional settings. opment and HIV/AIDS, Integrated Management of Childhood Illness, 2010. Disclosure [8] R. De La Fuente, W. Namkung, A. Mills, and A. S. Verkman, “Small-molecule screen identifes inhibitors of a human intesti- Tis manuscript is based on the thesis of the frst author. nal calcium-activated chloride channel,” Molecular Pharmacol- ogy,vol.73,no.3,pp.758–768,2008. Conflicts of Interest [9] L.Brunton,K.Parker,D.Blumenthal,andI.Buxton,“Treatment of disorders of bowel motility and water fux; ; Te authors declare no conficts of interest in this work. agents used in biliary and pancreatic disease,” in Goodman and Gilman’s Manual of Pharmacology and Terapeutics,pp.633– 652, McGraw-Hill, New York, NY, USA, 2008. Authors’ Contributions [10] H. L. DuPont, Z.-D. Jiang, C. D. Ericsson et al., “ versus ciprofoxacin for the treatment of traveler’s diarrhea: a All the authors participated in proposing the title, proposal randomized, double-blind clinical trial,” Clinical Infectious Dis- preparation, and writing up of the fnal report. Belay Mekon- eases,vol.33,no.11,pp.1807–1815,2001. nen conducted the laboratory activities, data compilation, [11] C. D. Ericsson, P. C. Johnson, H. L. Dupont, D. R. Mor- and analysis. Te manuscript was prepared by Assefa Belay gan, J. A. Bitsura, and F. J. de la Cabada, “Ciprofoxacin or Asrie. Finally, Belay Mekonnen and Zewdu Birhanu Wubneh trimethoprim-sulfamethoxazole as initial therapy for travelers’ reviewed the manuscript and some corrections were made diarrhea. A placebo-controlled, randomized trial,” Annals of basedontheircommentsbeforesubmission. Internal Medicine,vol.106,no.2,pp.216–220,1987. [12] A. C. Casburn-Jones and M. J. G. Farthing, “Management of Acknowledgments infectious diarrhoea,” Gut, vol. 53, no. 2, pp. 296–305, 2004. [13] D. R. Diniz-Santos, L. R. Silva, and N. Silva, “Antibiotics for the Te authors would like to acknowledge the University of empirical treatment of acute infectious diarrhea in children,” Gondar, Research and Community Service Core Process, for Te Brazilian Journal of Infectious Diseases,vol.10,no.3,pp. 217–227, 2006. providing the fnancial support to undertake this study as a thesis in partial fulfllment of the requirements for the M.S. [14] O. B. Balemba, Y. Bhattarai, C. Stenkamp-Strahm, M. S. B. Lesakit, and G. M. 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