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 Table of Contents  
ORIGINAL ARTICLE
Year : 2015  |  Volume : 8  |  Issue : 1  |  Page : 52-59

The effect of Nigella sativa aqueous extract on Dientamoeba fragilis: an in vivo experimental study


1 Department of Parasitology, Faculty of Medicine, Veterinary Medicine, Suez Canal University, Ismailia, Egypt
2 Department of Tropical Medicine, Faculty of Medicine, Veterinary Medicine, Suez Canal University, Ismailia, Egypt
3 Department of Pathology, Veterinary Medicine, Suez Canal University, Ismailia, Egypt

Date of Submission12-Jul-2014
Date of Acceptance24-Nov-2014
Date of Web Publication24-Aug-2015

Correspondence Address:
Omima M Eida
Assistant Professor of Parasitology, Department of Parasitology, Faculty of Medicine, Suez Canal University, Ismailia 41522
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1687-7942.163409

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  Abstract 

Background
Dientamoeba fragilis was considered as a commensal amoeba that inhabits the large intestine. Later, its association with irritable bowel syndrome drew attention to its pathogenicity. Metronidazole (MTZ) is the most recommended drug for treatment of D. fragilis and other pathogenic intestinal protozoa. Many studies reported that it is not suitable for children because of its mutagenicity and carcinogenic potential. However, still more work is needed to establish new, effective, and safe therapeutic agents against D. fragilis.
Aim of the work
The present study aimed at evaluating the effect of different doses of the aqueous extract of Nigella sativa on D. fragilis in experimentally infected mice in comparison with MTZ as a standard drug.
Materials and methods
Isolates of D. fragilis were obtained from patients complaining of acute/chronic intermittent diarrhea or diarrhea alternating with constipation with/without abdominal pain. Histopathological examination of cecal tissue of experimentally infected and treated mice with three different doses of N. sativa (125, 250, and 500 mg/kg/day) was compared with that of mice infected and treated by two doses of MTZ (62.5 and 125 mg/kg/day) as the standard treatment. Infected untreated mice were used as the control group.
Results
Histopathological examination of cecal tissue of the infected untreated group showed different degrees of pathological changes, which completely disappeared with the highest N. sativa dose (500 mg/kg). Concentrations below 500 mg/kg produced less severe pathological changes than in untreated animals. N. sativa in high dose significantly prevented cytopathic effect in mice 1 day after infection and for five consecutive days.
Conclusion
N. sativa has a potential therapeutic effect against D. fragilis infection in an experimental in vivo study. We recommend double-blind controlled clinical trials in humans to assess the use of N. sativa in management of human D. fragilis infection.

Keywords: Dientamoeba fragilis , histopathological assessment, in vivo, Nigella sativa


How to cite this article:
Eida OM, Eida AM, Eida MM, Dessouki AA. The effect of Nigella sativa aqueous extract on Dientamoeba fragilis: an in vivo experimental study. Parasitol United J 2015;8:52-9

How to cite this URL:
Eida OM, Eida AM, Eida MM, Dessouki AA. The effect of Nigella sativa aqueous extract on Dientamoeba fragilis: an in vivo experimental study. Parasitol United J [serial online] 2015 [cited 2017 Oct 22];8:52-9. Available from: http://www.new.puj.eg.net/text.asp?2015/8/1/52/163409


  Introduction Top


Dientamoeba fragilis is related to the trichomonad flagellates [1] . D. fragilis was described in 1918 as a binucleated, unflagellated protozoan that inhabits the large intestine [2] . On electron microscopy, it has been reclassified as an amoeba-flagellate rather than an amoeba [3] . According to molecular characterization of small subunit rDNA, ultrastructure, and antigenic analysis, it was considered a flagellate that lost its flagella and kintoplasts [4] .

D. fragilis was considered as a worldwide commensal organism with prevalence ranging between 0.5 and 42% [5] , proving in one study to be more prevalent than Giardia intestinalis [6] . More recent studies have shown that D. fragilis was diagnosed among patients with clinical manifestations in various areas of the world [7],[8],[9],[10] . However, many factors affect the prevalence of D. fragilis in fecal specimens, such as genetics, geographic location, population density, hygienic conditions, and methods of diagnosis. A high record of 52% was reported among children in the USA [11] as compared with 42% reported at a later time in Germany [12] . It was diagnosed in 14.2% of Egyptian patients with irritable bowel syndrome [13] . In Oman, Windsor et al. [14] considered D. fragilis the second most common pathogenic organism (5.1%) found in stool samples. Nonpathogenicity of D. fragilis was based on the fact that its source of nutrition relied on commensal bacteria in the gut rather than the tissues of the host [5] . More recent studies supported that D. fragilis is a pathogenic protozoan parasite and is a cause of abdominal pain, cramping, diarrhea, and vague abdominal complaints [7] . An analysis of D. fragilis ribosomal genes proved the existence of two genetically distinct pathogenic and nonpathogenic variants [15] . In a recent report from Egypt, PCR followed by RFLP analysis revealed the existence of two genetic variants among 25 D. fragilis isolates. Genotype I predominated in 23 (92%) samples, whereas the remaining two isolates corresponded to genotype II [16] .

Successful treatment of patients with irritable bowel syndrome diagnosed to have D. fragilis resulted in relief of symptoms, which added to the concept that D. fragilis is a pathogenic protozoan [6],[17] . D. fragilis trophozoites degenerate after few hours of stool collection and no cyst stage has been described [18] ; hence, diagnosis of D. fragilis depends on examination of fresh feces. In culture, voracious phagocytosis of rice starch distinguished D. fragilis from Blastocystis hominis, as the latter does not ingest these particles [5] . In an attempt to evaluate microscopy, culture, and PCR for detection of D. fragilis in stool samples, PCR detected 25 isolates (30.5%), modified Boeck Drbohlav's (MBD) culture detected 24 isolates (29.3%), whereas microscopy detected eight isolates (9.8%). Sensitivities of PCR, culture, and microscopy were 92.6, 88.9, and 29.6%, respectively [16] .

Metronidazole (MTZ) is the most common drug recommended for treatment of D. fragilis and other pathogenic intestinal protozoa [19] . However, many studies reported that it is not ideal for children because of its mutagenicity and carcinogenic potential [20],[21],[22] . Many side effects are also documented for other therapeutic agents used in treatment of D. fragilis, such as transient liver function abnormalities in patients treated with Diphetarsone [23] and deleterious effect on dental development in children treated with tetracycline. However, still more work is needed to establish new, effective, and safe therapeutic agents against D. fragilis. In the present study, we aimed to investigate the effect of Nigella sativa, a safe therapeutic herbal plant for treatment of D. fragilis. It is an herbaceous annual plant that belongs to the Family Ranunculacea and is commonly known as black seed, black cumin, or habitual Barakah. It grows in Mediterranean countries including Egypt, and the seeds have been documented for medical purposes since thousands of years ago [24] . The seeds have the same effect as MTZ in giardiasis [25] and have in vitro biocidal effects against all stages of Schistosoma mansoni and an inhibitory effect on its egg [26] . The black seed oil also showed promising prophylactic and therapeutic effects on murine toxoplasmosis [27] .

The aim of the present study was to evaluate the effect of different doses of the aqueous N. sativa extract on D. fragilis experimentally infected mice in comparison with mice treated with two doses of MTZ as a standard drug and mice treated with solvent of 20% gum acacia solution in distilled water as control.


  Patients and methods Top


Type of study

This was an experimental study.

Patients

A total of 250 patients complaining of acute or chronic intermittent diarrhea, or diarrhea alternating with constipation, with or without abdominal pain and attending Internal Medicine and Gastrointestinal Outpatient Clinics of Suez Canal University Hospitals (Ismailia, Egypt) were enrolled in the study. The study was conducted from June to December 2012. A questionnaire was fulfilled for every patient containing full history and clinical picture.

Stool examination and culture

Fresh stool specimens were collected and examined to exclude intestinal parasites by a wet smear stained with Lugol's iodine, followed by formalin ethyl acetate concentration technique [28] . Direct microscopic examination of fresh stool smears for D. fragilis stained with 10% Giemsa stain was performed immediately [28] . To exclude Cryptosporidium spp., Cyclospora spp., Isospora spp., and Microsporidium spp. infections, smears from preserved stool samples in 10% formalin were stained by modified acid-fast trichrome stain [28] . All of the fresh stool samples were also examined microscopically for pus cells and red blood cells and cultured for common enteric bacterial pathogens [29] . The fresh stool samples from 20 symptomatic patients positive only for D. fragilis were cultivated in the xenic MBD culture medium [30] . Culture was performed to increase the number of organisms to reach an adequate inoculating dose of D. fragilis, for inducing infection in mice. The culture was incubated at 37°C for 96 h. Each day, the sediment of the culture tubes was examined by light microscopy with ×40 objectives for trophozoites. Daily examination of culture for trophozoites was performed to count the number of D. fragilis by hemocytometer and adjust inoculating dose to 4 × 10 7 in 0.2-0.3 ml culture medium. This dose was used according to previous experience with successful experimental infection by B. hominis [31] .

Assessment of aqueous extracts of N. sativa medication

Sixty Swiss albino mice weighing between 25 and 35 g aged 4-6 weeks were used throughout the study. The mice were prepared for D. fragilis infection according to Ray and Chatterjee [32] . Briefly, 24 h before infection, the mice were starved and pretreated orally in the morning and evening with 0.5 ml 25% MgSO 4 in distilled water. On the next day, mice were orally inoculated with 4 × 10 7 of actively motile D. fragilis trophozoites in 0.2-0.3 ml culture medium, using a feeding tube [31] .

The mice were randomly divided into three groups. Group A included 10 infected nontreated mice, maintained as infection control. Group B included 20 mice infected and treated with MTZ as standard drug. They were subdivided into group BI, given a dose of 62.5 mg/kg/day, and group BII, given a dose of 125 mg/kg/day [33] . Group C was composed of 30 mice infected and treated with aqueous extracts of N. sativa. They were subdivided into group CI, given a dose of 125 mg/kg/day, group CII, given a dose of 250 mg/kg/day, and group CIII, given a dose of 500 mg/kg/day [19] . The infected control animals were given 0.2-0.3 ml of 20% gum acacia solution in distilled water to exclude the effect of the solvent.

Preparation of aqueous extracts

N. sativa seeds (250 g) were washed to remove any debris, dried, and then boiled in 1000 ml distilled water for 90 min and filtered through muslin. The final volume of the filtrate obtained was 320 ml. The filtrate was evaporated under reduced pressure and lyophilized [19] . Extract of N. sativa and of the standard drug, MTZ (Amriya Pharm. Ind. Alexandria, Egypt) in tablet form, was suspended in a solvent of 20% gum acacia solution in distilled water [33] . Stock solution of MTZ was prepared by dissolving 600 mg in 10 ml distilled water to give a final stock solution of 60 mg/ml. This was stored in the dark at 4°C [34] . All medications were administered daily orally using a feeding tube, for five consecutive days, beginning 24 h after infection with D. fragilis [33] . Stool elutes from mice in all groups were examined daily for D. fragilis trophozoite using Giemsa stain.

Histopathological studies

On the 60 th day, mice were killed by cervical dislocation and each cecum was longitudinally bisected. One-half of the cecum was placed in formalin fixative, cut into three to five equal cross-sections, paraffin embedded, and 4 µm sections were stained with H&E. Histopathological effect was scored blindly for each mouse. The numbers of histologically visible amoeba were scored 0-5 (0, none; 1, present but difficult to locate; 2, occasional, up to 10% of the lumen occupied by amoeba; 3, moderate, up to 25% of lumen occupied; 4, heavy, up to 50% of lumen occupied; 5, virtually complete occupation of the lumen by amoeba) [35] . Degree of inflammation was scored 0-4 (0, normal; 1, mucosal hyperplasia; 2, spotty infiltration by inflammatory cells not involving the entire thickness of the mucosa; 3, marked increase of inflammatory cells involving full thickness of mucosa; 4, marked increase of inflammatory cells in mucosa and submucosa, with tissue intact architecture) [35] .

Statistical study

The χ2 -test was used for association between two qualitatively expressed relationships. Significance levels were at P value less than 0.05. Statistical analysis was performed by SPSS, V. 16 (IBM company, USA).

Ethical considerations

Informed consents were taken from patients. Mice were purchased from the Faculty of Veterinary Medicine, Suez Canal University, and they were housed (five/cage) in proper room temperature and offered drinking water and regular mouse feed ad libitum.


  Results Top


In the present study, the total number of patients positive for D. fragilis was 92/250 (36.8%); 20 symptomatic patients (21.8%) had D. fragilis infection without concomitant infection, 72 (78.3%) had concomitant infection, of which 28 (30.4%) were symptomatic and 44 (47.8%) were asymptomatic. Stool samples from the symptomatic infected group without concomitant infection were cultured and inoculated in mice. This group was selected so that any pathological changes produced in mice would be due to D. fragilis infection alone. Among D. fragilis-infected patients (20), the most common symptoms were diarrhea, abdominal pain (50%), and flatus (30%), and other symptoms included vomiting, nausea, anorexia, and weight loss ([Table 1]).

N. sativa proved effective against D. fragilis in mice as evaluated by the high cure rate ([Table 2]) and the reduction of severity of large bowel inflammatory lesions in the treated groups compared with the untreated one ([Table 3]). The therapeutic effects of the two medications were apparently dose dependent.
Table 1 Concomitant infection among 28 symptomatic patients infected with D. fragilis

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Table 2 Effect of aqueous extracts of N. sativa on D. fragilis infection in mice compared with MTZ

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Table 3 Effect of aqueous extract of N. sativa compared with MTZ on histopathology of D. fragilis experimentally infected mice

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Histopathological sections of cecum from infected untreated mice (group A) showed severe crypt hyperplasia, congestion, and edema of the lamina propria of cecal mucosa, and the interglandular epithelial cells were sloughed into the lumen ([Figure 1] and [Figure 2]). Cecum from infected treated mice with low dose of MTZ (group BI) showed moderate to severe vacuolation of enterocytes ([Figure 3]), whereas that of group BII showed mild lymphocytic infiltration between cecal glands with degenerated trophozoites ([Figure 4]). Cecum from mice of group CI demonstrated that trophozoites were adherent to the surface epithelium or were trapped in the luminal mucus with absence of epithelial cell damage in some mice ([Figure 5]). Mononuclear inflammatory cellular infiltrate mainly lymphocytes were observed and some neutrophils were found in contact with trophozoites ([Figure 6]). Cecum of group CII showed mild hyperplasia of mucosa, mucinous degeneration along with mild mononuclear lymphocytic infiltrations with occasional to scarce numbers of degenerated trophozoites in the mucosa ([Figure 6]). Specimens from mice of group CIII showed normal mucosa with intact enterocytes denoting complete remission of infection after treatment by high dose of N. sativa ([Figure 7]). Estimation of D. fragilis and inflammation scores in all groups showed significant decrease in both D. fragilis trophozoites and inflammatory reaction in mice treated by N. sativa. There was no significant difference between effect of N. sativa (500 mg/kg/day) and MTZ (125 mg/kg/day).
Figure 1 Cecum of group A showing severe hyperplasia of mucosa (arrows) along with lymphocytic infiltrations [H and E (×400)] .

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Figure 2 Cecum of group A showing edema (E), congestion (C), and degenerated enterocytes [H and E (×200)] .

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Figure 3 Cecum of group BI showing moderate to severe vacuolation of enterocytes (arrows) [H and E (×200)] .

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Figure 4 Cecum of group BII showing mild lymphocytic infiltration between cecal glands with degenerated trophozoites (arrows) [H and E (×400)] .

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Figure 5 Cecum of group CI showing penetration of multiple D. fragilis trophozoites in cecal glands (arrows) [H and E (×400)].

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Figure 6 Cecum of group CII showing extension of inflammatory cells mainly lymphocytes and histocytes into the mucosa and submucosa (arrows) [H and E (×200)] .

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Figure 7 Cecum of group CIII showing complete remission of infection with normal intact enterocytes [H and E (×400)] .

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  Discussion Top


In the present study, the most common symptoms associated with D. fragilis infection were abdominal pain, diarrhea, flatus, and vomiting. These results were in accordance with numerous reports that showed an association between D. fragilis infection and clinical symptoms, principally abdominal pain, diarrhea, nausea, vomiting, and fatigue [13],[36],[37],[38] .

In the present study, we successfully used the xenic MBD culture medium to grow D. fragilis for infecting the experimental animals and adjusted the proper inoculating dose of D. fragilis that induced pathogenicity in mice. The proper dose was similar to that used by Munasinghe et al. [18] . The absence of other concomitant pathogenic parasites in the stools of the group of symptomatic patients solely infected with D. fragilis supports its pathogenicity. According to Johnson et al. [5] , D. fragilis isolation was technically limited by inability to maintain this organism in axenic cultures. Accordingly, drug susceptibility testing in culture needs to be performed in the presence of supporting bacteria, which would make it very difficult to determine whether the studied antimicrobial agent was active against D. fragilis or the bacteria supporting its survival.

Besides using N. sativa as flavoring agent and spice for some special baked products, it is also used as carminative and diuretic [19] . Other beneficial pharmacological effects have been attributed to various crude and purified components of black seeds, including antihistaminergic, antihypertensive, hypoglycemic, antimicrobial, mast cell stabilizing, and anti-inflammatory activities [39] . These include immune stimulation, anti-inflammatory [40] , antitumor [41] , and antioxidant [42] . Most of these biological activities have been attributed to thymoquinone, the main active constituent of the volatile oil extracted from the seeds [43] .

The present study demonstrated that N. sativa is effective against D. fragilis in vivo. The effects of the two tested compounds were dose dependent. In the present study, mice treated with MTZ at a concentration of 125 mg/kg/day for 5 days were successfully cured (100%) of D. fragilis infection. This confirms sensitivity of the parasite to this dose; the lower dose of 62.5 mg/kg/day gave only 60% cure. Our results on efficacy of MTZ were similar to studies by other investigators [44],[45] . In contrast to our results, Norberg et al. [46] found that the curative effect of MTZ was moderate, after using it for treatment of 32 patients infected with D. fragilis. The drug was given at various dosages and for various lengths of time, but no correlation was found between dosage or length of treatment and clinical success. Four patients recovered after the MTZ treatment, whereas 12 patients had recurring or reduced symptoms. N. sativa extract appeared to be most effective at a concentration of 500 mg/kg/day, as this dose cleared all D. fragilis from the intestine of mice on the sixth day of examination. This is comparable with MTZ at the dose of 125 mg/kg/day on the same day of examination. However, treatment with N. sativa extract at a concentration of less than 500 mg/kg/day did not cure all mice, and there was mucosal hyperplasia indicating the lower effectiveness of the extract at lower concentrations. The use of N. sativa extract to treat D. fragilis infection at least helped in reducing severity of infection. All infected untreated mice were positive for D. fragilis at the time of sacrifice except one mouse, which may have been due to low inoculating dose of D. fragilis in culture media or high immune response of the mouse, which cleared the infection. Several researchers studied the effect of N. sativa on the immune system and reported an increase in the ratio of helper to suppressor T cells and enhancement of natural killer cell activity in normal volunteers [47] . In vitro studies confirmed that N. sativa enhanced the production of interleukin-3 by human lymphocytes and had a stimulatory effect on macrophages [48] . Besides, the immunomodulatory effect of purified proteins of N. sativa was studied in mixed lymphocyte cultures and was proved to cause changes in the levels of cytokines with an enhancing effect on production of interleukin-1b and tumor necrosis factor-α [49] . In in vitro experiments, both interferon-g and tumor necrosis factor-α induce production of reactive nitrogen intermediates including nitric oxide, which is an important regulatory molecule involved in the minimization of the immunopathological alterations induced by different parasites [49] .

Our results were in accordance with other studies conducted on different protozoan parasites. Bishara and Masoud [25] proved that the alcoholic extract of N. sativa was as effective as MTZ in experimental giardiasis. A study conducted by El Wakil [19] also reported the inhibitory effect of N. sativa on two B. hominis isolates in vitro, whereas Rayan et al. [27] concluded that oil extracted from the black seed showed promising prophylactic and therapeutic effects on murine toxoplasmosis. Although N. sativa aqueous extract had the lowest effect on Trichomonas vaginalis growth in vitro compared with MTZ and wheat germ agglutinin, producing a lethal result only after 48 h, it still had a remarkable effect [50] . The authors reported that wheat germ agglutinin and N. sativa aqueous extract remarkably inhibited the motility of T. vaginalis trophozoites in vitro. The results showed a promising effect in using wheat germ agglutinin and N. sativa aqueous extract in treating T. vaginalis infection [50] . Our results were also in accordance with those of El-Shenawy et al. [26] who proved that N. sativa oil prevented most of the hematological and biochemical changes and markedly improved the antioxidant capacity of schistosomiasis-infected and treated mice compared with the infected and untreated ones.


  Conclusion Top


N. sativa has a potential therapeutic effect against D. fragilis infection in experimental mice especially when using a high dose. It is available, of popular use as spice, at low cost and has no side effects. All of these characteristics encourage its use as treatment for D. fragilis infection. It is recommended to perform double-blind controlled clinical trials in human to assess N. sativa as treatment for D. fragilis infection.


  Acknowledgements Top


The authors acknowledge Dr. Ahamed Ali, PhD in Microbiology for his assistance in the microbiological cultures. In addition, many thanks to Dr. Walaa El-Dakroury, Pharmacist in holding company for biological material and vaccine (VACSERA) for her help in preparation of N. sativa. They also thank Dr. Hala Abdel Hameed, Assistant Professor of Special Education, Suez Canal University, for performing statistics of present work.


  Author contribution Top


O.M. Eida provided the original idea of the research and planned the study design. She shared in the experimental studies , analyzed the results, and wrote the manuscript. A.M. Eida shared in the study design, the experimental studies and manuscript writing. M.M. Eida selected the cases and shared in manuscript writing. A.A. Dessouki shared in the experimental studies and assessed the histopathological results.

Conflicts of interest

There are no conflicts of interest.

 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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Abstract
Introduction
Patients and methods
Results
Discussion
Conclusion
Acknowledgements
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References
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