In vivo anti-diarrheal activity of jujube honey on castor oil-induced diarrhea in mice

Abdelouafi Benmouloud

Abdelouafi Benmouloud,Department of Biology,University of M'hamed Bougara Boumerdes (UMBB),Boumerdes 35000,Algeria;Biology and Physiology of Organism Department,Houari Boumedienne University of Sciences and Technology (USTHB),Arid Area Research Laboratory,Bab Ezzouar 16111,Algiers;University of Algiers 1 Benyoucef Benkhedda,Department of Nature and Life Sciences,Algiers 16000,Algeria

Abstract OBJECTIVE:To evaluate the antidiarrheal effect of jujube honey on experimentally castor oil-induced diarrhea in mice by using different testing models(diarrhea,enteropooling and gastrointestinal motility).METHODS:The mice intragastric administration castor oil was post-treated after 30 min with jujube honey,diluted jujube honey and loperamide or vehicles in different experimental groups.The onset and number of wet defecation on the absorbent paper was recorded for each animal for 4 h.Plasma was examined for C-reactive protein (CRP) and nitric oxide(NO)for clinical inflammation evaluation.The oxidative stress was investigated by superoxide dismutase(SOD)and catalase levels.RESULTS:The diluted jujube honey exhibited an important antidiarrheal activity manifested by significant delay in onset of diarrhea(P<0.05),loss in number of wet stools (P <0.001),total number of stools(P<0.001)and total stool weight of fecal output(P<0.001)in 4 h in castor oil-induced diarrheal groups.The inhibition of intestinal transit of charcoal meal(P<0.01)is a most likely mechanism that may account for antidiarrheal effect of jujube honey.It also significantly increased SOD and catalase levels (P <0.001) and significantly decreased CRP and NO in plasma diarrheic mice(P<0.01).CONCLUSION:The results of this research reveal that the jujube honey contains pharmacologically active compounds with antidiarrheal properties.However,diluted honey is more effective in the treatment of diarrhea and imbalance of intestinal motility.In conclusion,these findings illustrated the antidiarrheal activity effect of jujube honey and which has the strongest evidence supporting its use in the treatment of diarrhea in traditional medicine.

Keywords:diarrhea;Jujube honey;castor oil;intestinal motility;mice

INTRODUCTION

Diarrheal diseases were the third leading cause of death of children younger than 5 in 2017 and are becoming an increasing burden in people aged 70 and over.In 2017,1.6 million people died because of diarrheal diseases.1Diarrhea,a common gastrointestinal disorder characterized by an increased frequency of bowel movements,wet stool and abdominal pains,is a serious public health challenge.2,3Diarrhea can be triggered by many different factors,such as infections,food intolerance,intestinal disorders,among others.4,5The precise mechanism is unknown,yet alteration of intestinal transit or the intestinal microflora has been proposed.6The use of synthetic drugs such as diphenoxylate,atropine maleate,kaolin,pectin,antibiotics,and oral rehydration solutions in the treatment of diarrhea is often associated with many problems such as accessibility,high cost of drugs and the multiresistance of microorganisms.7Diarrhea and gastroenteritis are found to resolve quickly with honey.8Clinical studies have similarly shown an improvement of honey in the treatment of infantile gastroenteritis,with a decrease in the duration of diarrhea,being also useful in the recovery of hydration post-gastroenteritis.9Honey has been suggested as potentially useful for various conditions of the gastrointestinal tract,such as periodontal and other oral disorders,dyspepsia and as part of oral rehydration therapy.10In addition,honey may be effective as a part of oral rehydration therapy,and as a clinical trial,honey shows therapeutic effects in the treatment of infants and children admitted into hospital with gastroenteritis indicated remarkable reduced duration of diarrhea in the honey-treated patients.11,12It is also considered as one of the potential candidates for treatment of diarrhea because it contains a natural combination of probiotics,prebiotics and zinc.13It has been shown that honey reduces prostaglandin levels and increases nitric oxide levels.14Honey is also one of the most widely natural dietary antioxidant whose components are responsible for the redox properties are likely to be flavonoids,phenolic acids,enzymes,vitamins and minerals.15

Interestingly,the jujube honey is characterized by high pH,electrical conductivity,potassium,calcium,high phenolic content,medium flavonoid content and other molecules.16,17Jujube honey is a unifloral honey derived from the Jujube lotus tree,Ziziphus lotus (L.) Lam.,commonly called"Sedra or Sidr"belonging to the family Rhamnaceae occurring across the Mediterranean basin,north Africa and the Sahara and the Arabian peninsula.18,17Recently it was suggested that therapeutic effect and antioxidant activity properties of honey increased with the solubility of its bioactive compounds in water.19-21In view of the fact that honey has various biological activities,this study aimed to evaluate the therapeutic potential of jujube honey on its anti-diarrheic effects against castor oil-induced diarrhea in mice.The study also investigated the efficacy of diluted honey and we hypothesized that it would be more effective than non-diluted in the treatment of diarrheal diseases.

MATERIALS AND METHODS

Honey sample and chemical agents

Jujube honey (Sidr Honey),was collected from beekeepers in Laghou at region (Algeria) during summer season.The honey was stored in hermetically closed glass jars in dark at－4 ℃.Loperamide (Dimépra®20 mg) was obtained from Pharmalliance Laboratories(Algiers,Algeria)to use as a reference standard medication for diarrhea;charcoal meal(Eucarbon®)from Laboratoire F.TRENKA Chemisch-Pharmazeutische Fabrik(Vienna,Austria);castor oil from Tiralab Pharmaceuticals (Algiers,Algeria).All remaining chemicals used in the current study were of the highest quality and analytical grade.

Experimental animals

Forty-eight male balb/c mice (Pasteur Institute,Algiers City,Algeria),6 weeks old,weighing between 25 and 30 g were used in this study.The animals were housed in the animal facility at Faculty of Science,University M'hamed BOUGARA,Boumerdes,Algeria and acclimated for one week under the environmental conditions prior to the study(12 h light and dark cycle at an ambient temperature of(25±1)℃and relative humidity of 50%-60%).The animals had free access to commercial standard pellet diet and water ad libitum.The use of experimental animals was conducted in strict compliance with the rules and regulations established by the Research Ethics Committee of the Algerian Association of Animal Experimental Sciences (45/DGLPAG/DVA.SDA.14).

Activity of jujube honey on castor oil-induced diarrhea

The method described by Awouterset al22was followed for this study.Four groups of six mice each were fasted for 18 h with free access to water.To induce diarrhea,castor oil (0.5 mL) was orally administered to all mice.Thirty minutes after castor oil administration,the first group (diarrheal control) received distilled water (0.5 mL),the second group received the reference drug,loperamide (5 mg/kg bw),while the other two groups received one of the jujube honey doses:5 or 2.5 g/kg bw by oral route,while the lowest dose was obtained by dilution in a volume of 1∶1 with sterilized distilled water.After these treatments,each mouse was individually placed in a cage lined with previously weighed filter paper.These filter papers were changed every thirty minutes for 4 h.23Absence of diarrheal dropping was recorded as a positive result,indicating protection.During each 30 min,stool mass,stool count and the number of diarrheal stool wet feces were recorded.The percentage inhibition of diarrhea,total defecation and that of faecal output were calculated using the following formula:

Activity of jujube honey on small Intestinal transit

Four groups of six mice each were fasted for 18 h with free access to water.The first group received distilled water (0.5 mL),the second group received loperamide(5 mg/kg bw),while the other two groups received one of the jujube honey doses:5 or 2.5 g/kg bw by oral route.Thirty minutes after these treatments,each mouse received 0.5 mL of castor oil-induced intestinal motility test.Again,after an hour,1 mL of 5% charcoal suspension was given orally to each animal for gastrointestinal motility test.Then,all animals were euthanized after 30 min by cervical dislocation under 25%urethane anesthesia.The blood samples were collected in heparinized tubes for hematological and biochemical analyses.Blood plasma was obtained by centrifugation at 3000 rpm for 10 min at 4 ℃and then analyzed within 24 h.The abdomen of each animal was also opened and the intestine was removed.The total distance of the small intestine and the distance traveled by the charcoal meal in the intestine were measured and then expressed as a percentage of the total distance traveled from the pylorus to the caecum.24The peristaltic index and percentage of inhibition were also calculated by using the formula described below.

Activity of jujube honey on castor oil-induced enteropooling

Intraluminal fluid accumulation was carried out according to the method of Robertet al.25Four groups of six mice each were deprived of food for 18 h while water was allowed ad libitum.Then,the animals were treated in the same manner as described above.One hour later,each group of animals received orally 0.5 mL of castor oil.The mice were euthanized under urethane anesthesia 1 hour after castor oil administration and the small intestine removed and weighed with its content.The small intestinal contents were collected by milking into a graduated tube,its volume measured and the emptied small intestine reweighed.24Then,percent reductions in the weight of intestinal content and volume of intestinal content,relative to the negative control group,were calculated using the following formulas:

Measurement of antioxidant capacity

Superoxide dismutase (SOD) activity was measured by the nitroblue tetrazolium (NBT) reduction method.26The superoxide anion radical (O2•−) generated by the catalytic reaction between xanthine and xanthine oxidase reduces NBT to blue formazan.5 µL of plasma was added to 2 mL of enzyme working solution for 10 min and the absorbance was then measured at 560 nm using a spectrophotometer.One unit of SOD activity was defined as the amount of enzyme activity that results in 50% inhibition of NBT to formazan.Catalase (CAT) activity was measured as the rate of H2O2reduction at 37 ℃at 240 nm using a spectrophotometer based on the method of Aebiet al.27CAT catalyzes the oxidation of H2O2to H2O and O2.When H2O2is in excess,the surplus H2O2can oxidize the chromogenic substrate to form a red product via peroxidase.The assay mixture consisted of 50µL of 1 M Tris(Tris-hydroxymethyl-aminomethane)-HCl buffer containing 5 mM ethylene diamine tetraacetic acid (pH 7.4),900 µL of 10 mM H2O2,30 µL of deionized water,and 20µL of the plasma.One unit of CAT activity is the amount of enzyme that catalyzes 1 µmoL H2O2in 1 min at 25 ℃and pH 7.0.

C-Reactive Protein assay

In order to study the inflammatory status,C-Reactive Protein (CRP) plasma concentration was determined by Spinreact kit according to the manufacturer's instructions (Spinreact,S.A./S.A.U.Ctra.Santa Coloma,Spain),and the values were expressed asµg/L.

Analysis of leukocyte counts

Whole blood sample was analyzed for the counts of total white blood cells using the routine hematology laboratory method (Abacus 380 Automated Hematology Analyzer,Diatron,Budapest,Hungary).

Measurement of plasma NO level

The plasma NO level was measured using the modified Griess method.28Briefly,50 µL of plasma sample was added to 50 µL Griess reagent [N-(1-naphthyl)-ethylene diamine dihydrochloride (0.1%),1%sulfanilamide and 2.5%H3PO4]and mixed.After incubation at 37 ℃for 60 min,the nitrite levels were determined spectrophotometrically at 543 nm and the nitrite concentration was determined by comparing the sample absorbance to a standard curve for sodium nitrite.The results were expressed asµmole/L.

Statistical analysis

The results were expressed as mean ± standard error of the mean (SEM).The differences between means were determined by Student'st-test for unpaired samples or analysis of variance (ANOVA) followed by Bonferroni test.Statistical analysis was performed using GraphPad Prism 7.0 (GraphPad Software Inc.,La Jolla,CA,USA).The level of significance for rejection of the null hypothesis was set at 5%(P<0.05).

RESULTS

Effect of jujube honey on castor oil-induced diarrhea

The jujube honey treatment was found to be effective against castor oil-induced diarrhea in terms of decrease of frequency of defecation and number of wet feces.The onset of diarrhea induced by castor oil was significantly delayed (P <0.001) in loperamide group(128.000 ± 2.966) min when compared with the distilled water-treated group (89.500 ± 2.929) min (Table 1).A significant decrease in the number of defecation instances within 4 hours was observed with all jujube honey-treated groups compared with the negative control group.The mean total number of defecation instances in jujube honey groups was 5.875 ± 0.426 and 3.101 ± 0.224 at 5 and 2.5 g/kg doses,respectively,while this value was 10.358 ± 0.484 in the negative control group.The percent inhibition of total defecation relative to the negative control group was 43.28 and 65.76%at 5 g/kg dose of jujube honey and standard drug,respectively.The diluted jujube honey at 2.5g/kg dose had shown a more marked reduction in defecation (70.04%) relative to the vehicle-treated group(Table 1).

The mean number of diarrheal drops in the jujube honey-treated groups was 0.323 ± 0.022 and 0.255 ±0.011 at 5 and 2.5 g/kg doses,respectively,while in the loperamide treated group it was 0.206 ± 0.012.Percentage of faecal output was also reduced by diluted jujube honey and loperamide,in which the lowest dose(2.5 g/kg)producing a better effect compared to jujube honey group at 5 g/kg dose.Number of wet feces was reduced with percentage inhibition of 65.14% (P <0.001) with jujube honey at dose of 5 g/kg.The diluted jujube honey has also shown significant reduction(P <0.001) of the number of wet defecations at dose of 2.5 g/kg with highest percentage inhibition of 83.46% compared to jujube honey group.Loperamide treated group (positive control) has also shown highly significant reduction (P <0.001) of wet defecation with percentage inhibition of 78.02%(Table 1).

Effect of jujube honey on castor oil-Induced enteropooling

Compared to control groups,treatment with jujube honey inhibited castor oil-induced enteropooling in mice as revealed by reduction in weight and volume of intestinal contents.The mean weights of the intestinal content were (0.338 ± 0.017) and (0.123 ± 0.013) g,while the mean volumes of the intestinal content were(0.260 ± 0.013) and (0.094 ± 0.005) mL,respectively,at 5 and 2.5 g/kg doses of jujube honey.The jujube honey treatment decreased the mean weight of the intestinal content by 53.73%and 75.23%at 5 g/kg dose of jujube honey and loperamide,respectively,compared to the negative controls.The highest percent inhibition of 82.71%was scored by diluted jujube honey(Table 2).

The jujube honey treatment at dose of 5 g/kg significantly (P <0.001),reduced the volume of fluid accumulation in the intestine with percentage inhibition of 52% relative to the negative control group.The standard drug loperamide 5 mg/kg significantly (P <0.001) reduced the volume of intestinal fluid produced by castor oil with percentage inhibition 74.15% relative to the distilled water-treated group.The diluted jujube honey at dose 2.5 g/kg had better effect than the standard drug with percentage inhibition of 82.58%relative to negative control group(Table 2).

Effect of jujube honey on the intestinal transit of charcoal meal

The effect of the jujube honey treatment on the intestinal motility is shown in Table 3.The jujube honey at 5 and 2.5 g/kg oral doses exhibited significant inhibition of intestinal motility with 24.35%and 35.45%,respectively.However,the standard drug loperamide produced significant inhibition(37.76%)in intestinal motility relative to the control group.

Figure 1A shows the progress of charcoal meal in isolated mouse intestinal tracts after intragastric administration of castor oil and different treatments.The peristalsis index for distilled water-treated animals was 71.0%± 6.0% while jujube honey (5 and 2.5 g/kg) delayed charcoal movement and had peristalsis indices of 52.5% ± 3.2% and 44.0% ± 2.9% respectively.The standard drug loperamide (5 mg/kg) caused a peristalsis index of 44.4%±2.2%and had almost similar peristalsis index with diluted jujube honey at dose 2.5 g/kg.Figure 1B compares the peristalsis indexes from the different treatments.

Figure 1 Effect of jujube honey on the intestinal motility in castor oil induced diarrhea in mice

Effect of jujube honey on plasma Superoxide Dismutase(SOD)and catalase activities,CRP and NO in castor oil-induced diarrhea

The results of the enzymatic antioxidant analyses are shown in Table 4.Treatment of mice with jujube honey caused a significant increase in both SOD(54.67%)and catalase activities (32.60%) as compared to the control group(P<0.05).Whereas,pretreatment of castor oil-induced diarrhea with both loperamide and diluted jujube honey has reported high significant increases in plasma SOD (73.39% and 92.66%;P <0.01,respectively) and catalase enzyme activity(44.13% and 52.61 %;P <0.01,respectively) compared to the control group.

Table 3 Effect of jujube honey on castor oil induced gastrointestinal transit in mice

The results of CRP assay using as inflammatory biomarker are shown also in Table 4.Treatment of mice with jujube honey caused a significant decrease in plasma CRP levels (－15.42%;P <0.05) as compared to the control group.Pretreatment of castor oil-induced diarrhea with diluted jujube honey caused a high significant decrease (－22.91%;P <0.01) as compared to the control group.Treatment of mice with jujube honey caused a significant decrease in NO plasma level(－27.74%) as compared to the control group (P <0.01) (Table 4).Although pretreatment with both diluted jujube honey and loperamide reveal high significant decrease in plasma NO levels (－58.51% and－52.11%;P<0.01,respectively).

Effect of jujube honey on white blood cells count in castor oil-induced diarrhea

The changes of total white blood cells count (WBC)were shown in Table 4.Compared with control group,a significant increase in the level of WBC (17.2%;P <0.01)was observed in diluted jujube honey-treated animals after castor oil-induced diarrhea.However,pretreatment of castor oil-induced diarrhea mice with raw jujube honey or loperamide caused a no significant increase of WBC (P>0.05) as compared to the control group.

Table 4 Effect of jujube honey on plasma SOD,and catalase activities,CRP,WBC and NO in castor oil-induced diarrhea

DISCUSSION

The present study aimed to evaluate the antidiarrheal effect of jujube honey on experimentally induced diarrhea in mice by using different testing models (diarrhea,enteropooling and gastrointestinal motility).In all testing models,diarrhea was induced by administering castor oil to animals.Castor oil has been largely used for induction of diarrhea due to its active metabolite,a ricinoleic acid which is released by the action of lipases in the upper part of the small intestine.29Ricinoleic acid mediates its action by nitric monoxide30and also by binding to prostaglandin E receptor 3 on smooth muscle cells31and it initiates diarrhea via mechanisms such as irritation of gastrointestinal mucosa,electrolyte secretion and electrolyte absorption decrease from the intestine and colon.32

As shown in results,the jujube honey treatment reduced the total fecal output and diarrheal drops in castor oil-treated mice compared with control group.The percent reduction in the frequency of defecation and diarrhea was increased with a corresponding decrease in the dose of jujube honey.The percent inhibition produced by the lowest dose of jujube honey was closer to the inhibition produced by loperamide.This finding may indicate that the jujube honey treatment at relatively low dose has a significant antisecretory effect and antidiarrheal action in castor oil-induced diarrhea model.The results obtained from this model are in line with reports elsewhere,where composite raw honey of Bangladesh at dose(250 mg/kg),showed significant inhibition of diarrhea in castor oil-induced diarrhea in mice.33

In castor oil-induced enteropooling test,the jujube honey treatment showed a significant reduction in the intraluminal fluid content when compared to control group.The percent reduction in weight of intestinal content was increased with diluted jujube honey.The results in this model revealed that the effect of the lowest dose of the jujube honey on intestinal fluid accumulation was found to be closer to loperamide.

The diluted jujube honey effect was similar to loperamide,which is one of the most widely employed drugs against diarrheal diseases;as shown in other studies that loperamide effectively antagonised diarrhea induced by castor oil.34,35So,this result indicated that diluted jujube honey is a promising candidate for new antidiarrheal drug development.

Secondary metabolites such as flavonoids36,37are reported to possess anti-diarrheal activity due to their ability to inhibit intestinal motility.It's known that flavonoids inhibit the release of prostaglandins;thereby inhibit secretion induced by castor oil and facilitate absorption of electrolytes.23,38Therefore,the anti-enteropooling activity may be due to the presence of bioactive compounds in particularly flavonoids in jujube honey.Ingeneral,flavonoids have been reported to have several pharmacological activities,including antidiarrheal activity,which has been attributed to antisecretory and antimicrobial action effects.39Phytochemical screening of jujube honey performed by Zerrouket al.17revealed presence of flavonoids and phenolic acids.

In the gastrointestinal transit model,the charcoal meal was used to follow the intestinal content transit because the reduction of gastrointestinal motility is one mechanism by which many antidiarrheal agents can act on intestine.40The reduction in gastrointestinal motility increases the time of stay of gastrointestinal contents in the intestine and this may promote intestinal water and electrolyte absorption.41Thus,in the intestinal propulsive movement decrease in the charcoal meal model is further supported by the antimotility property of jujube honey.

Reduced in CRP plasma level was related to the anti-inflammatory effect of jujube honey.Kassimet al42demonstrated that Gelam honey has an inhibitory effect on prostaglandin E2 thereby reducing pain and oedema in the inflammatory tissues in rats.This suggestion is validated by the facts that castor oil induced diarrhea is related to stimulation of prostaglandins biosynthesis.25,43It was reported that ingestion of diluted natural honey has produced reductions on concentrations of prostaglandins such as prostaglandin E2,prostaglandin F2a and thromboxane B2 in plasma of normal individuals,thus showing antiinflammatory effects.44Thus,the antidiarrheal action exerted by jujube honey may also be associated with the inhibition of prostaglandin formation.

It is widely known that the activation of NF-κB plays a key role in the pathogenesis of inflammation.45Therefore,Gelam honey has just been documented to inhibit the inflammatory process by inhibiting NF-κB pathway.46In the present study also we suggest that nitric oxide may partially contribute to the antidiarrheal activity of jujube honey.

The antidiarrheal effects of Jujube honey may also be related to its potent antioxidant potential as observed in this study by the increase of the antioxidant activity of catalase and SOD enzymes.The antioxidant action and free radical scavengers of Jujube honey may be related to its antioxidant phenolic and flavonoid contents.16,17The antioxidant activities of flavonoids involve reactive oxygen species scavenging,transition metal ion chelation,increase of enzymatic and non-enzymatic antioxidants and reduction of lipid peroxidation.47We have found that diluted honey was more effective in diarrheal disease treatment.This finding was comparable to that demonstrated in some clinical studies using diluted honey in oral rehydration solution in infants and children with gastroenteritis.9,20

The molecular mechanism of jujube honey effect on secretory diarrhea still remains elusive.The chloride channels,as the last step in Cl−secretion,have become attractive targets for antisecretory therapy.48Chloride secretion across the intestinal epithelia occurs mainly through Cl−channels including the cystic fibrosis transmembrane conductance regulator (CFTR),Ca2+-activated Cl−channels (CaCCs) and chloride channel-2(ClC-2),among which CFTR and CaCC may play pivotal roles.49,50In the intestine,CFTR,a cAMP-activated chloride channel,is mainly expressed in the crypt.51Natural products have been identified with antidiarrheal efficacy in humans and a putative mechanism of action involving Cl−channel inhibition.52It has been well documented that flavonoid (including quercetin) can activate CFTR chloride channel.53The jujube honey may have inhibited the secretion of water into the lumen by blocking this mechanismviaflavonoids and particularly the quercetin.

Interestingly,the identification of CaCC chloride channels as new molecular targets of quercetin would highlight the possibility of different types of CaCC proteins in the intestine.Inhibition of Anoctamin-1/CaCC may delay the movement of the intestine and thus increase the fluid absorption time.54The ability of quercetin to delay intestinal peristalsis could be of great use to the treatment of intestinal dynamic disorder.CaCC activation can be acquired by improving the cytoplasmic Ca2+concentration.55Some investigators believe that chloride secretion by quercetin is due to the activation of CFTR and basolateral Na+-K+-2Cl-cotransporter.56CFTR and CaCCs chloride ion channels have become important targets for the treatment of intestinal imbalance in recent years.50

The possible mechanisms of antidiarrheal activity of jujube honey could be related to inhibition of secretion,reducing intraluminal fluid accumulation or enhancing water and electrolyte absorption.However,the isolation and characterization of jujube honey bioactive agents as well as the exact mechanism of actions need to be investigated in further studies.

In conclusion,jujube honey likely exerts its antidiarrheal effect by improvement of antioxidant activities and anti-inflammatory properties via its potent pharmacologically active compounds.Therefore,this study provides a scientific support for the traditional medicine use of jujube honey for the treatment of diarrheal diseases.