Jfae(food & health-parta) vol3-1 (2005)

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Science and Technology
Meri-Rastilantie 3 B, FI-00980 Journal of Food, Agriculture & Environment V ol.9 (2): 10-17. 2011 Helsinki, Finland e-mail: [email protected] Anti-coccidial activity of Nigella sativa L.
Hanadi B. Baghdadi and Ebtisam M. Al-Mathal *
Department of Biology, College of Science, Dammam University, Dammam 31311, Saudi Arabia. Received 20 November 2010, accepted 29 March 2011. The objective of the present study was to evaluate in vivo the anti-coccidial effect of Nigella sativa L. seeds in aqueous suspensions and oil emulsions in rabbits. Both treatments had anti-coccidial effects as demonstrated by reduced fecal oocyst shedding, significant body weight gain, recovery of normal liver weights and remarkable improvement of liver tissue histopathology. This improvements includes disappearance of bleeding between hepatic lobules and severe reduction in the infiltration of the inflammatory cells in the portal area, reduction in the various stages of parasites in the bile ducts, return of the natural hypatocytes radial arrangement, disappearance of all severe symptoms and the heading of the tissue structure towards full recuperation. Though both treatments were effective, the N. sativa oil emulsion treatments showed more rapid and robust anti-parasitic effects than did the water suspension treatments. As previously indicated, N. sativa treatment did not cause any harmful side effects, though uninfected animals that received N. sativa treatments gained more weight than did the untreated controls. Therefore, N. sativa seed suspensions and oil emulsions are safe and effective anti-parasitic treatments for Eimeria stiedae infection in rabbits. Future studies focus on determining the active components of N. sativa seeds, optimizing the effective doses and elucidating the mechanism of action. Key words: Hepatic coccidiosis, Nigella sativa, fecal oocysts counting, body weight, histopathological study.
Materials and Methods
Due to the continual emergence of drug resistant parasites and Preparation of Eimeria stiedae oocysts: E. stiedae oocysts were
the increasing incidence of animal condemnation due to coccidian collected from liver nodules and gallbladder lumens of naturally- infection, novel approaches are urgently needed to reduce infected rabbits. Oocysts were washed and concentrated using economic losses. Nigella sativa (family: Ranunculaceae), also the flotation method 30, sporulated and stored in 2.5% potassium known as black cumin, is a herbaceous annual plant used in dichromate at 4°C. traditional medicine for thousands of years because of its anti- convulsive and antioxidant effects 1-7. Many pharmacological Plant materials: Water suspensions and oil emulsions of Nigella
investigations suggest that N. sativa has anti-diabetic effects 8-12, sativa L. seeds were used in this study produced by Pharco therapeutic effects due to its anti-inflammatory 13, antioxidant 11 Pharmaceutical Company (Egypt, Alexandria). Emulsions were and anti-tumor 14-17 effects. Several studies have also indicated its prepared using Cremophore EL as an emulsifying agent 31.
strong preventive effect against hepatocarcinogenesis 18-21.
Though numerous studies have been conducted to determine Animals: A total of 80 male New Zealand white rabbits, aged 3-4
the effects of N. sativa seeds on numerous diseases and microbes, months and weighing 1.2-2.0 kg, were bred at the King Saud only a few studies have examined their effect on parasitic infections. University Research Center. Rabbits were free from coccidian Most of these studies examined only the anti-schistosomal infection as determined by coccidian-free fecal samples over 3 activities of N. sativa. These studies indicated that treatment with consecutive days using the concentration flotation technique. N. sativa reduces the rate of parasitic egg lying and improves The rabbits were weighed and randomly distributed into 8 groups liver functions of the infected animals 22-24. Additional studies of 10 rabbits each. Rabbits were separately housed in wire-floored indicated that N. sativa is effective against infection with batteries under sanitary conditions with controlled humidity, trematodes, cestodes and nematodes 25-27.
temperature, light periods (12-h light/12-h dark cycles) and fed Eimeria stiedae is one of the primary parasites with severe Purina chow diet and water ad libitum. infectivity in rabbits and can cause hepatic coccidiosis. E. stiedaeinfection also affects body weight and feeding rates that can result Experimental design: Rabbits were administered a single dose
in the death of infected rabbits, especially young animals 28, 29.
of 1 × 103 sporulated oocysts through a gastric tube 32 for all This study was undertaken to investigate, in vivo, the anti-coccidial experiments. On day 14 post-inoculation (pi), rabbits were treated effect of N. sativa seeds in water suspensions and oil emulsions with distilled water, N. sativa suspensions, or N. sativa oil by examining the liver tissue structure of rabbits infected with E. emulsions. One hour before feeding, each rabbit was given a dose of 400 mg of Nigella sativa L. seeds in water suspensions or oil Journal of Food, Agriculture & Environment, Vol.9 (2), April 2011 emulsions per kilogram of body weight 8 orally every morning Table 1. Mean number of oocysts/g feces (mean ± SE ) in
after an overnight fast for 14 consecutive days. infected and infected and treated groups. Groups Description Uninfected and untreated; Control group Infected and untreated 76.42 37142.857 73.86 Uninfected and given distilled water Uninfected and treated with N. sativa suspension Infected and treated with N. sativa suspension Uninfected and given emulsifying agent Uninfected and treated with N. sativa oil emulsion opg before treatment – opg after treatment Opg before treatment Infected and treated with N. sativa oil emulsion Effect of treatments on body weight: The infected rabbits that
Fecal analysis: Fecal samples were collected and examined
did not receive treatments (G2) continuously lost weight starting on day 7 pi until day 14 pi, when the oocysts appeared in throughout the course of the experiment (Table 2; P:0.01) as the feces and the rabbits were given treatments. Fecal oocysts compared to G1. The infected animals that received treatment were concentrated using the flotation technique and counted using similarly lost weight through day 14 pi, but then had remarkable a hemocytometer 33 on days 7, 14, 21 and 28 pi. The reduction (%) significant weight gain by day 21 pi, 7 days after treatment began, of oocysts was determined based on the number of oocysts per that continued through day 28 pi. gram of feces before and after treatment. By day 28 pi, the weight of the infected animals treated with the N. sativa oil emulsion (G8) returned to the same level as the animals Body weight: Animals were weighed at the beginning of the
in the control group (G1) with no significant differences. Though experiment and on days 7, 14, 21 and 28 pi. the animals that received the N. sativa suspension (G5) gained weight, they did not return to the same weight as the control Histopathological analysis: Livers were weighed after they were
group. There was a significant difference (P:0.01) between the removed from euthanized rabbits on days 14 and 28 pi. The relative body weights of G5 and G8 animals on day 28 pi. Uninfected liver weight for each rabbit was calculated as the percentage of animals that received N. sativa treatments (G4 and G7) also gained the total body weight 34. Liver tissues were fixed in 10% formalin, significantly weight (P:0.05), comparing with animals in (G1) which dehydrated with ethanol, cleared and embedded in paraffin wax. did not gain significantly weight over the course of the experiment. Sections 2- to 3-mm thick were cut and stained with haematoxylin Similar to the infected animals, the animals that received the N. and eosin (H&E) 35.
sativa oil emulsion (G7) gained more weight than those that received the suspension (G4). Statistical analysis: Data were analyzed using the SPSS package
program (SPSS 8.0, 15.0 Windows). Data are represented as
Liver morphological changes: The mean liver weights and the
arithmetic means with standard errors. One-way analysis of relative liver weights of the infected and untreated rabbits (G2) variance and t-tests were performed to demonstrate significant were significantly higher (P:0.01) than the uninfected and differences between groups. Values of P<0.05 were considered untreated rabbits (G1) throughout the duration of the experiment (Table 3). The liver weights were not significantly different between the infected groups (G2, G5 and G8) on day 14 pi. However, following N. sativa treatment, liver weights drastically and Infection and behavioral observations: Fecal examination showed
significantly decreased in the G5 and G8 animals as compared that oocysts were present on day 14 pi in all infected groups (G2, with the untreated animals on day 28 pi (P:0.01). The average G5 and G8). At day 28 pi, infected rabbits treated with the N. liver weights of the uninfected animals were not affected by N. sativa suspension (G5) or oil emulsion (G8) recovered their sativa treatment. appetite, started to gain weight, became active again and recovered from diarrhea with feces returning to its beady form. Histopathological analysis of liver tissue: Normal tissue structure
Treated rabbits in G4,G6 and G7showed normal appetite and was present in liver samples from uninfected rabbits (G1, G2, G4, behavior throughout all experiment period. G6 and G7), regardless of treatments, on day 14 and 28 pi (Fig. 1A- D). Classical hepatic coccidiosis pathological changes in liver Effect of treatments on fecal oocyst levels: The levels of fecal
tissue of infected rabbits (G2) were present on day 14 (Fig. 2A-D) oocysts were not significantly different between the three infected and 28 pi (Fig. 3A-D). Similar pathological changes were present groups on day 14 pi (Table 1; P:0.10). Infected rabbits that in liver tissues from the infected rabbits (G5 and G8) on day 14 pi. received the N. sativa treatments (G5 and G8) showed reduced Following treatment with N. sativa, visible improvement was fecal oocyst output on day 21 pi (7 days post-treatment) as present in the liver tissue of the infected rabbits on day 28 pi. compared to the untreated infected animals in G2 (P:0.01), with Though the hepatocytes retained their radial shape, the central decreases of 76.42% and 73.86%, respectively. Furthermore, fecal vein of the hepatic lobules became less congested (Fig. 4A-D). oocysts were undetected on day 28 pi in G8 rabbits and on day 35 Additionally, decreases were seen in the bleeding between the pi in G5 rabbits. hepatic lobules, fatty degeneration, infiltration of inflammatory cells into the portal area and the various stages of parasites in the Journal of Food, Agriculture & Environment, Vol.9 (2), April 2011 bile ducts at day 28 pi in G2. These improvements indicated that the tissues were making full recoveries following treatment with N. sativa.
Though both N. sativa treatments induced improved liver tissue health, the N. sativa oil emulsion (G8) was more effective than the water suspension (G5) as shown in Fig. 5A-D. The liver tissue from G8 rabbits on day 28 pi indicated that the hepatocytes had recovered their radial arrangements, central veins had incomplete healing of the endothelial lining encircling it, the bleeding in between hepatic lobules had almost disappeared, and the various stages of parasites had disappeared completely. This study indicates the effectiveness of N. sativa treatment for the treatment of coccidiosis in rabbits. The dose administered in the study (400 mg/kg of body weight) is considered by researchers to be an average dose and does not have any identified side effects 8. Previous studies have used similar doses (250-500 mg/ kg of body weight) to protect the liver from carbon tetrachloride toxicity 36. Furthermore, doses as high as 2 g of N. sativa per kilogram of body weight proved to be safe and highly effective for reducing the rate of glucose absorption in rats with experimental diabetes 12. N. sativa treatments (0.2, 40 and 100 mg/ kg of body weight) were effective in treating schistosomiasis as it decreased the number of oocysts and reduced liver damage 22, 37, . Mohamed et al. 23 indicated that low concentrations of N. sativa (10-110 ppm) reduced the number of eggs produced by female parasites. A previous study indicated that N. sativa oil had anti- parasitic effects in that it helped stop bleeding in patients infected with Schistosoma sp. worms 38. In this study, disease symptoms improved and body weights increased within 2 weeks of treatment with the N. sativa seed suspensions and emulsions, indicating recuperation from E. stiedae infection 32, 39-47.
Animals treated with N. sativa seed suspensions or oil emulsions gained weight following treatment whether or not they were infected with E. stiedae. The uninfected animals that received N. sativa treatments (G4 and G7) showed significant weight gains on days 21 and 28 as compared to the control animals (G1). N. sativa seeds contain appetizing materials that may increase food intake and that contain concentrated weight-increasing fatty materials 48. In both the infected (G5 and G8) and uninfected (G4 and G7) groups, the animals that received the N. sativa oil emulsions gained weight more rapidly. Specifically, animals in the G8 group increased in weight by 45.20% as compared to infected and untreated animals (G2), whereas G5 animals increased in weight by only 30% on day 28 pi. This difference in weight gain was very significant between the two treated groups and may be due to the higher percentage of fatty materials in the emulsion 48.
Since the coccidial parasite is intracellular, a degree of lipid solubility may be important for achieving higher intracellular The disappearance of oocysts from the feces of groups G5 and G8 may be because N. sativa seeds contain powerful antioxidants and are considered good immunity boosters 1-7. Therefore, the antioxidants found in N. sativa seeds could limit the growth and development of parasites in the host body and reduce the oocyte Mean body weight (mean ± SE) of rabbits in all groups. formation and appearance in feces 22. Disappearance or reduction . Relative liver weight of rabbits in all groups. of fecal oocyst shedding is considered an indicator of recuperation. Treatment of E. stiedae infections with N. sativa seeds in water Relative liver weight = Journal of Food, Agriculture & Environment, Vol.9 (2), April 2011 suspensions or oil emulsions was highly effective at reducing Ghannadi, A., Hajhashemi, V. and Jafarabadi, H. 2005. An investigation of the analgesic and anti-inflammatory effects of Nigella sativa seed relative liver weights. This may be due to the concurrent decreases polyphenols. J. Med. Food. 8(4):488-493.
in liver weights and increases in body weight. Furthermore, rabbits 4Ilhan, A., Gurel, A., Armutcu, F., Kamisli, S. and Iraz, M. 2005. infected with E. stiedae showed remarkable improvement in liver Antiepileptogenic antioxidant, effects of Nigella satica oil against tissue histopathology on day 28 pi following 14 days of treatment pentylenetecrazol - induced kindling in mice. Neuropharmacol. with N. sativa (G5 and G8). Most disease symptoms disappeared and the liver recovered its normal shape. In addition, there was a 5Vahdati-Mashhadian, N., Rakhshandeh, H. and Omidi, A. 2005. An huge reduction in the various stages of parasites in the tissue and investigation on LD and subacute hepatic toxicity of Nigella sativa immature oocysts in the bile ducts, resulting in reduced fecal seed extracts in mice. Pharmazie 60(7):544-547.
oocyst shedding and improved external symptoms. These findings Kacem, R. and Meraihi, Z. 2006. Effects of essential oil extracted from Nigella sativa L. seeds and its main components on human neutrophil support previous studies indicating that the use of N. sativa seed elastase activity. Yakugaku Zasshi 126(4):301-305.
oil is very effective in reducing the effect of Schistosoma mansoni 7Rahman, M. M., Garvey, M., Piddock, L. J. and Gibbons, S. 2008. worms on the liver and for renewing hepatocytes 26, 37, 38.
Antibacterial terpenes from the oleo-resin of Commiphora molmol Though both treatments showed beneficial effects, the N. sativa (Engl.). Phytother. Res. 22(10):1356–1360.
oil emulsion was more effective in all aspects examined than was 8Farah, K. M., Aloji, Y., Shimizu, Y., Shiina, T., Nikami, H. and Takewaki, the water suspension in these short-term experiments. The T. 2004. Mechanisms of the hypoglycaemic and immunopotentiating effective material in the N. sativa oil emulsion may have been effects of Nigella sativa L. oil in streptozotocin induced diabetic more concentrated than in the crude seed suspension, therefore hamsters. Res. Vet. Sci. 77(2):123-129.
having a more rapid and robust effect. The N. sativa oil emulsion Kanter, M., Coskun, O., Korkmaz, A. and Oters, S. 2004. Effect of Nigella sativa on oxidative in stress and beta-cell damage in contains higher concentrations of alkaloid nigellicine, which has stareptozocin-induced diabetic rats. Anat. Rec. A. Discov. Mol. Cell a deadly effect on parasites 38. In addition, N. sativa oil extracts Evol. Biol. 279(1):685-691.
hamper the production of leukotrienes that cause damage to cells 10Rchid, H., Chevassus, H., Nmila, R., Guiral, C., Petit, P., Chokairi, M. and tissues 48. The effectiveness of the N. sativa treatments may and Sauvaire, Y. 2004. Nigella sativa seed extracts enhance glucose– be increased because the seeds contained thymoquinone that induced insulin release from rat isolated langerhans islats. Fundam.
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and leakage of enzymes from the cells 49, 22.
Kaleem, M., Kirmani, D., Asif, M., Ahmed, Q. and Bano, B. 2006. This study did not identify any harmful side effects of the N. Biochemical effects of Nigella sativa L. seeds in diabetic rats. Ind. J. sativa treatments. The uninfected animals (G4, G6 and G7) were in Exp. Biol. 44(9):745-748.
12Meddah, B., Ducroc, R., Faouzi, M. E. A., Eto, B., Mahraoui, L., good condition and did not suffer any harmful side effects. The Andaloussi, A. B., Martineau, L. C., Cherrah, Y. and Haddad, P. S. only difference between the uninfected animals was a significant 2009. Nigella sativa inhibits intestinal glucose absorption and improves increase in body weight of the treated animals (G4 and G7) as glucose tolerance in rats. J. Ethnopharmacol. 121:419-424.
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9.45%), likely due to a higher percentage of fatty materials in the Salomi, M. J., Nair, S. C. and Panikkar, K. R. 1991. Inhibitory effects emulsion. Furthermore, this study did not identify any negative of Nigella sativa and saffron (Crocus sativus) on chemical carcinogenesis
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Journal of Food, Agriculture & Environment, Vol.9 (2), April 2011

Figure 1. A: Photomicrograph of transverse section (T. S.) in rabbit liver tissue (G1) on day 14 pi. showing a normal central vein (Cv) lined by a
simple squamous epithelium that is continuous with the endothelial lining of the hepatic sinusoids (S) and hepatic cords (Hc) composed of
hepatocytes. H&E staining (original magnification, 1000×). B: Photomicrograph of T. S. in rabbit liver tissue (G1) on day 14 pi. showing a normal
portal area containing portal vein (Pv), an artery (A), bile duct (Bd), and lymph visual (Lv),. H&E staining (original magnification, 1500×). C:
Photomicrograph of (T. S.) in rabbit liver tissue (G1) on day 28 pi. showing normal central vein (. H&E staining (original magnification, 1000×). D:
Photomicrograph of T. S. in rabbit liver tissue (G1) on day 28 pi showing a normal portal area (original magnification, 1000×).
Figure 2. A: Photomicrograph of T. S. in rabbit liver tissue (G2) on day 14 pi. showing a dilated congested central vein with ruptured endothelial lining
and hemorrhagic areas. H&E staining (original magnification, 750×). B: Photomicrograph of T. S. in rabbit liver tissue (G2) on day 14 pi. showing a
sever hemorrhagic sinusoids and fatty degeneration. H&E staining (original magnification, 1000×). C: Photomicrograph of T. S. in rabbit liver tissue
(G2) on day 14 pi. showing hyperplasia and hypertrophy of the bile duct epithelium. H&E staining (original magnification, 1000×). D: Photomicrograph
of T. S. in rabbit liver tissue (G2) on day 14 pi. showing the bile duct containing different development stages of E.stiedae surrounded by
inflammatory reaction and fibrosis. H&E staining (original magnification, 875×).
Journal of Food, Agriculture & Environment, Vol.9 (2), April 2011 Figure 3. A: Photomicrograph of T. S. in rabbit liver tissue (G2) on day 28 pi. showing a dilated congested central vein with severe ruptured endothelial lining
and hemorrhagic areas, sinusoidal dilation and atrophy of cords. H&E staining (original magnification, 1000×). B: Photomicrograph of T. S. in rabbit liver tissue
(G2) on day 28 pi. showing hydropic degeneration of the hepatocytes and sever hemorrhagic sinusoids. H&E staining (original magnification, 1000×). C:
Photomicrograph of T. S. in rabbit liver tissue (G2) on day 28 pi. showing papilliform projections of epithelium into the lumen of bile duct containing very large
number of developed oocysts. H&E staining (original magnification, 300×). D: Photomicrograph of T. S. in rabbit liver tissue (G2) on day 28 pi. showing
different development stages of E.stiedae the lumen of bile duct. H&E staining (original magnification, 1400×).
Figure 4. A: Photomicrograph of T. S. in rabbit liver tissue (G5) on day 28 pi. showing healing of the congested central vein, incomplete healing of the
endothelial lining and the hepatocytes encircling the central vein. H&E staining (original magnification, 1000×). B: Photomicrograph of T. S. in rabbit liver
tissue (G5) on day 28 pi. showing healing of the hemorrhagic tissue between sinusoids, and disappearance of fatty degeneration. H&E staining (original
magnification, 750×). C: Photomicrograph of T. S. in rabbit liver tissue (G5) on day 28 pi. showing a large reduction in lymphocyte infiltration in the portal
area. H&E staining (original magnification, 1000×). D: Photomicrograph of T. S. in rabbit liver tissue (G5) on day 28 pi. showing a large reduction in the
parasite stages. H&E staining (original magnification, 1166.6×).
Journal of Food, Agriculture & Environment, Vol.9 (2), April 2011 Figure 5. A: Photomicrograph of T. S. in rabbit liver tissue (G8) on day 28 pi. showing healing of hemorrhagic tissue and almost normal endothelial
lining and hepatocytes encircling the central vein. The hepatocytes laminate regained their original radial shape. H&E staining (original magnification,
750×). B: Photomicrograph of T. S. in rabbit liver tissue (G8) on day 28 pi. showing almost normal sinusoids, and fatty vacuoles disappeared. H&E
staining (original magnification, 750×). C: Photomicrograph of T. S. in rabbit liver tissue (G8) on day 28 pi. showing a large reduction in lymphocyte
infiltration. H&E staining (original magnification, 1000×). D: Photomicrograph of T. S. in rabbit liver tissue (G8) on day 28 days pi. showing a almost
disappearance of the parasite stages from the lumen of the bile duct. H&E staining (original magnification, 1500×).
Journal of Food, Agriculture & Environment, Vol.9 (2), April 2011

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