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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 35  |  Issue : 3  |  Page : 307-311

Effect of Nigella sativa oil on Schistosoma mansoni immature worms in experimentally infected mice


Department of Parasitology, Benha Faculty of Medicine, Benha University, Egypt

Date of Submission03-Jan-2018
Date of Acceptance30-Jan-2018
Date of Web Publication07-Jan-2019

Correspondence Address:
Dr. Ghada H Omar
Parasitology Department, Faculty of Medecine, Benha University, Qulyobia Governorate, 13518
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/bmfj.bmfj_2_18

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  Abstract 


Background Nigella sativa oil is one of the promising drugs of a plant origin that have an antischistosomal effect.
Aim The aim of the present work was to explore the role of N. sativa oil either alone or in combination with praziquantel on immature worms of Schistosoma mansoni.
Materials and methods N. sativa oil capsules dissolved in corn oil were administered to S. mansoni-infected mice aiming to study the therapeutic and prophylactic effects. The study included six groups of mice: (a) noninfected and nontreated, (b) infected and nontreated, (c) prophylactic N. sativa oil group, (d) praziquantel group, (e) therapeutic N. sativa oil group, and (d) combined group. All mice were killed 4 weeks after infection and treatment. The antischistosomal effect of N. sativa oil was assessed by worm burden, histopathology, and scanning electron microscopy.
Results Therapeutic N. sativa oil group showed the highest significant decrease in the mean number of immature worm burden (R% was 57%) when given after infection followed by the combined group (R%=55.67%), whereas prophylactic N. sativa oil group showed nonsignificant decrease (R%=37%). The least changes were observed in praziquantel group (R%=0%).
Conclusion N. sativa oil has a potent effect on immature S. mansoni which could be helpful for potentiating praziquantel effect and thus reducing development of resistance.

Keywords: immature worms, Nigella sativa oil, Schistosoma mansoni


How to cite this article:
Al Hamshary AS, Nagati IM, Eraky MA, Abou-Ouf EA, Kholy AA, Omar GH. Effect of Nigella sativa oil on Schistosoma mansoni immature worms in experimentally infected mice. Benha Med J 2018;35:307-11

How to cite this URL:
Al Hamshary AS, Nagati IM, Eraky MA, Abou-Ouf EA, Kholy AA, Omar GH. Effect of Nigella sativa oil on Schistosoma mansoni immature worms in experimentally infected mice. Benha Med J [serial online] 2018 [cited 2019 Dec 15];35:307-11. Available from: http://www.bmfj.eg.net/text.asp?2018/35/3/307/249421




  Introduction Top


Schistosomiasis is a chronic and debilitating disease that threatens millions of people, particularly those living in the rural poor areas in the developing world [1]. Currently, the foremost drug used for the treatment of schistosomiasis is praziquantel [2]. Referring to the possibility of appearance of drug-resistant parasites, especially with retreatment regimens in endemic areas, search for new schistosomicidal drug is essential [3].

The minor activity of praziquantel against immature schistosomes is believed to be a clue factor explaining failure of treatment in areas extremely endemic for bilharziasis [4]. Nowadays, there is a great awareness of the therapeutic value of natural products and medicinal plants that are frequently considered to be less toxic and free of adverse effects than synthetic drugs in treating some diseases [5]. Among Muslims, the seeds of the miracle herb, Nigella sativa, are considered the remedy for all diseases except death in one of the prophetic hadith [6]. The most important active compound of N. sativa seeds is thymoquinone [7]. N. sativa has a wide range of pharmacological activities including anti-inflammatory, anticancer, antioxidant, immunomodulatory, antimicrobial, and wound healing effects [8]. Moreover, it has antiparasitic effects against some protozoal infections such as Entamoeba histolytica trophozoites [9], toxoplasmosis [10], and Trichomonas vaginalis [11]. It has anticestodal and antinematodal properties [12]. On that basis, antischistosomal activity of N. sativa has been investigated.

The aim of the present study was to emphasize on the effective role of N. sativa oil as either therapeutic, prophylactic, or in combination with praziquantel in the treatment of immature worms of Schistosoma mansoni.


  Materials and methods Top


Herbal extract

N. sativa oil was obtained as soft gelatin capsules (450 mg) (Pharco Pharmaceutical, Alexandria, Egypt). Capsules were opened, and oil was dissolved in corn oil [13] and orally administered to mice at a dose given of 1.14 g/kg body weight [14].

Praziquantel (Alexandria Company for Pharmaceuticals and Chemical Industries) was freshly suspended in 13 ml of 2% Cremophore (El Sigma Chemical Co., St. Louis, MO, USA) and orally administered to mice at a dose given of 500 mg/kg body weight for 2 consecutive days [15].

Parasites and animals

Cercariae of S. mansoni were obtained from infected Biomphalaria alexandrina snails, which were reared and maintained at Schistosome Biological Supply Program, Theodor Bilharz Research Institute, Giza, Egypt. A total of 60 laboratory-bred male Swiss albino mice, CD1 bred, were used in this study. All mice were infected with 60 S. mansoni cercariae suspended in 0.2 ml water through subcutaneous injection.

Experimental design

Mice were divided into six groups, with 10 mice each, as follows: noninfected and nontreated, infected and nontreated, prophylactic N. sativa oil group [treated with N. sativa oil (1.14 g/kg) daily for 2 weeks before infection], praziquantel group (500 mg/kg) for 2 successive days 3 weeks after infection), therapeutic N. sativa oil group [treated with N. sativa oil (1.14 g /kg) daily for 2 weeks] starting from the second day after infection, and combined group (treated by N. sativa oil and praziquantel after infection).

All mice were killed 4 weeks after infection.

Evaluation of drug efficacy

Worm burden

Immature schistosoma worms recovery was assessed by portomesenteric perfusion technique 4 weeks after infection, according to the method of Duvall and De Witt [16].

Histopathological examination for liver specimens

Mice livers were fixed for 48 h in 10% buffered formalin and then embedded in paraffin. Five sections (5 μ in thickness) were taken from each liver specimen, with each section being at a distance of at least 500 μm from the preceding one [17]. Sections were stained with hematoxylin and eosin [18].

Scanning electron microscopy examination of immature worms

In an attempt to determine whether N. sativa oil can affect morphological alterations in the recovered S. mansoni, immature worms collected from all groups were prepared for examination using scanning electron microscopy (SEM). Worms were thoroughly washed in PBS, then fixed in 3% glutaraldehyde in PBS at room temperature for 24 h. Following fixation, worms were dehydrated and critically point dried. Dehydrated worms were mounted on stainless steel holders, sputtcoated with gold, and examined using JEM-1200 scanning electron microscope (JEOL USA, Inc., MA, USA), fitted with a camera. Areas in the worms that showed specific changes were examined and photographed; these were mainly suckers and tubercles on the tegument [19].

Statistical analysis

The data were recorded on an ‘investigation report form’. These data were tabulated, coded, and then analyzed using the computer program SPSS version 16. Analysis of variance test was used to compare between more than two groups of numerical (parametric) data and Kruskal–Wallis test between more than two groups of numerical (nonparametric) data. Student’s t-test was used to compare between mean of two groups of numerical (parametric) data. For continuous nonparametric data, Mann–Whitney U-test was used for inter-group analysis. P value less than 0.05 was considered statistically significant, and a P value less than 0.01 was considered highly significant in all analyses.


  Results Top


Worm burden

Our results showed that N. sativa oil had the highest significant decrease in the mean number of immature worm burden (R% was 57%) when given after infection followed by the combined group (R% was 55.67%), whereas the prophylactic group treated with N. sativa oil showed nonsignificant decrease in the mean number of immature worm burden (R% was 37%). The least changes in immature worm burden were observed in praziquantel-treated group (R% was 0%). There was a statistically high significant difference seen in N. sativa oil treated group and combined group compared with the control group (P=0.003), whereas there was an insignificant difference seen in prophylactic group and praziquantel group compared with the control group ([Table 1]).
Table 1 Effect of Nigella sativa oil, PZQ, and combination of both drugs on Schistosoma mansoni immature worm burden in the infected mice

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Histopathologial study

Liver sections obtained from mice of the therapeutic N. sativa oil group that were killed 4 weeks after infection showed improvement of hepatic pathology in the form of less hydrobic degeneration and less inflammatory infilterate with moderately dilated sinusoids that were lined by von Kupffer cells ([Figure 1]), whereas the combined group showed no improvement of hepatic pathology in the form of more hydrobic degeneration and more inflammatory infilterate mainly lymphocytes with dilated sinusoids that were lined by hyperpigmented von Kupffer cells ([Figure 2]). Prohylactic N. sativa oil group and praziquantel group showed no improvement of hepatic pathology.
Figure 1 Therapeutic Nigella sativa group: liver sections of S.mansoni infected mice showing improvement of hydropic degeneration of the hepatocytes (red arrow) hyperplastic pigmented Von kupffer cells (yellow arrow). (H& E stain) (×400).

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Figure 2 Combined group: liver sections of S.mansoni infected mice showing no improvement of hydropic degeneration of the hepatocytes (red arrow) hyperplastic pigmented Von kupffer cells (yellow arrow). (H& E stain) (×400).

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Scanning electron microscope

The highest effect on immature worms was noticed in the therapeutic N. sativa oil group, where swelling and obliteration of the oral sucker, tegumental destruction with vesicle formation, and severe peeling were seen ([Figure 3]). Combined group showed erosions, blebbing of male tegument with constriction of its body, and destruction of female tegument ([Figure 4]). Prophylactic group showed shrunken both oral and ventral suckers, and the male and female tegument shows wrinkles, peeling, and erosions. However, the praziquantel group had no effect on immature schistosoma worms.
Figure 3 Therapeutic Nigella sativa group: Scanning electron microscopy (SEM) of Schistosoma mansoni immature male worm showing oral sucker (OS), tegumental destruction (TD), vesicle (v) and peeling (P). (×250).

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Figure 4 Combined group: Scanning electron microscopy (SEM) of Schistosoma mansoni immature couple showing an oral sucker (OS), ventral sucker (VS), constriction of body (Co), erosions (E), blebing (Bl) and tegument destruction of female (×60).

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  Discussion Top


The development of resistance toward many antiparasitic drugs is an important topic of concern. The search for natural products, as alternative, has received great attention [20]. According to our results presented in [Table 1], the therapeutic N. sativa oil group shows a high significant decrease in the mean number of immature worms when given after infection either alone (R% was 57%) or in combination with praziquantel (R% was 55.67%), whereas the prophylactic N. sativa oil group shows nonsignificant decrease in the mean number of immature worm burden (R% was 37%). However, praziquantel had no effect on immature worm burden. These findings are in parallel with those reported by Saada et al. [21] who reported that praziquantel is unable to treat the migrating juvenile stages. However, Shaw [22] revealed that praziquantel caused only moderate damage to both the tegument and subtegumental tissues in juvenile worms, and Aragon et al. [23] confirmed that 6 weeks after infection, schistosomes are sensitive to praziquantel, whereas 4 weeks after infection, schistosomes are not.

According to our results, liver sections obtained from mice of the therapeutic N. sativa oil group that were killed 4 weeks after infection showed improvement of hepatic pathology in the form of less hydrobic degeneration and less inflammatory infilterate with moderately dilated sinusoids that were lined by von Kupffer cells. However, Sheir et al. [24] reported that noninfected mice treated with N. sativa oil showed abnormal liver cells with a pyknotic nuclei and a cytoplasmic vacuolization. According to our results, liver sections of S. mansoni-infected mice treated with praziquantel showed scattered inflammatory cells, mainly lymphocytes, hydropic degeneration of the hepatocytes, and dilated sinusoids that were lined by hyperplastic pigmented von Kupffer cells. Similar results were obtained by Sheir et al. [24] who stated that treatment with praziquantel in noninfected mice showed abnormal hepatocytes with dilated sinusoidal spaces. Regarding combined group and prophylactic N. sativa oil group, both of them showed no improvement of hepatic pathology in the form of more hydrobic degeneration and more inflammatory infilterate mainly lymphocytes with dilated sinusoids that were lined by hyperpigmented von Kupffer cells.

SEM plays an important role in elucidating the detailed morphology and different alterations of the S. mansoni tegument allowing the interpretation of its functionality [25]. The current study is the first to document the morphological alterations of immature S. mansoni worms following N. sativa oil treatment in vivo by using SEM.

Therapeutic effect of N. sativa oil on immature worms was evident, and SEM of juvenile worms showed swelling and obliteration of the oral sucker, extensive tegumental destruction, edema, vesicles formation, multiple erosions, and peeling.

This is explained by Kusel et al. [26] who reported that the function of glycoproteins in the parasite surface is to act as an immunochemical barrier against the host’s immune system. Therefore, the tegumental alteration in S. mansoni worms after treatment with black seed oil could have exerted an intense effect on the metabolic activities of the parasite and in turn expose the tegument to the host’s immune attack [27]. Regarding the effect of combined therapy on immature worms, it showed constriction of the male body, erosions, blebbing of male tegument, and destruction of female tegument. These findings emphasize the additional effect of N. sativa oil on praziquantel in treatment of immature worms, thus reducing the expected resistance against praziquantel.

SEM of S. mansoni immature worms obtained from mice infected and received prophylactic treatment with N. sativa oil showed shrinking of both oral and ventral suckers; the male and female tegument showed wrinkles, peeling, and erosions; the female body showed bending and twisting.

SEM of S. mansoni immature worms obtained from mice treated with praziquantel showed normal both oral sucker and ventral sucker and no effect on tegument. However, Kamel and Bayaumy [28] stated that tegumental alterations were observed on S. mansoni immature worms after 24 h of incubation with a concentration of 5 μg/ml praziquantel (PZQ), in the form of contraction of the worm body with vesicles and focal lesions in the middle region of the worm. However, Pica-Mattoccia et al. [29] reported that PZQ had little effect on juvenile worms at the concentrations of 0.1 and 1 μg/ml.

In conclusion, this study showed that N. sativa oil has a potent effect on immature S. mansoni which could be helpful for potentiating praziquantel effect and thus reducing development of resistance. The efficacy of Ni. sativa oil is dependent on time of starting of the treatment, as therapeutic treatment showed more effectiveness than prophylactic treatment.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4]
 
 
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