A Study on the Amoebicidal Effect of Nigella Sativa Aqueous and Alcoholic Extracts and Wheat Germ Agglutinin on Pathogenic Acanthamoeba

Background: Acanthamoeba keratitis is a corneal disease associated predominantly with contact lens wear. Treatment of Acanthamoeba keratitis has been fairly successful using chlorhexidine but may be accompanied by drug toxicity and development of resistance. Medicinal plants can be an alternative resource of novel anti-protozoal drugs with high effectiveness and low toxicity. Nigella sativa seeds are considered one of the potential natural sources in folk medicine. Wheat germ agglutinin (WGA) has shown therapeutic effects against protozoa.

Objective: is to study the in vitro effect of Nigella sativa aqueous and alcoholic extracts as well as wheat germ agglutinin on pathogenic Acanthamoeba spp. in comparison to chlorhexidine 0.02%.

Methods: Collection of corneal samples for isolation of Acanthamoebae, preparation of non-nutrient agar-Escherichia coli plates for cultivation of the obtained samples,microscopic examination of the plates for detection and identification of Acanthamoeba growth daily for two weeks, preparation of the study medications (Nigella sativa aqueous and alcoholic extracts as well as WGA). Preparation of chlorhexidine 0.02% as a drug control. Evaluation of the amoebicidal effect of different concentrations of N. Sativa aqueous extract, N. sativa alcoholic extract and WGA in comparison to chlorhexidine 0.02%.Determining the parasite inhibition percentage and the minimal lethal concentration (MLC) of each medication.

Results: The MLC of N. sativa aqueous extracton Acanthamoeba trophozoites was 5 mg/ml after 24 h incubation and 500 μg/ml after 48 hrs. The drug control (chlorhexidine 0.02%) had an inhibition percentage of 100% at incubation durations of 24, 48 and 72 hrs. The MLC of N. sativa aqueous extract on Acanthamoeba cysts was 30 mg/ml after 24 hrs and 25 mg/ml after 48 hrs with a highly significant (p<0.001) and a significant (p<0.05) differences respectively compared to the drug control. Chlorhexidine 0.02% had an inhibition percentage of 56% after 24 hrs, 64% after 48 hrs and 80% after 72 hrs incubation. The MLC of N. sativa alcoholic extract on Acanthamoeba trophozoites was 10 mg/ml after 24 hr incubation and 500 μg/ ml after 48 hrs. The MLC of N. sativa alcoholic extract on Acanthamoeba cysts was 30 mg/ml after 24 hrs incubation with a highly significant difference (p<0.001) compared to the drug control. The MLC of WGA on Acanthamoeba trophozoites was 50 μg /ml after 24, 48 and 72 hrs incubation which was similar to the drug control. Wheat germ agglutinin concentration of 1 mg/ ml caused inhibition of Acanthamoeba cysts with percentages of 60% and 68% after 24 and 48 hrsincubation respectively which were higher than those caused by the drug control (56% after 24 hrs and 64% after 48 hrs) with statistically insignificant difference (p>0.05). Inhibition percentage of 80% was obtained by concentration of 2 mg/ml after 72 hrs incubation which was similar to the drug control. The same inhibition percentage (80%) was also obtained by the same concentration after 48 hrs incubation which was significantly higher (p<0.05) than the drug control (64%).

Conclusion: N. sativa aqueous and alcoholic extracts as well as WGA showed that these agents had considerable lethal effects on Acanthamoeba trophozoites and cysts. These effects were found to be dose and time dependent. They were also found comparable to or even superior than the effect of the commonly used chlorhexidine (0.02%) as a chemotherapeutic.

Author(s): Khadiga Ahmed Ismail, Azza Abdel Salam El kadery, Nadia Mohammad Sabry and Omnia Sobhi Mohammad

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