Keywords

Acanthamoebae spp; Nigella sativa; Wheat germ agglutinin

Introduction

Acanthamoebae are ubiquitous potentially pathogenic freeliving amoebae that occur worldwide in soil and water. They are tolerant of a wide range of osmolarity, enabling them to survive in distilled water, tissue culture media, mammalian body fluids and sea water [1].

Acanthamoeba keratitis is a corneal disease associated predominantly with contact lens wear. The occurrence of Acanthamoeba keratitis has been rising since 1990 in correlation to the increasing number of contact lens wearers [2].

Treatment of Acanthamoeba keratitis has been fairly successful using a variety of drugs. Brolene, a commercially available eye medication containing propamidine isethionate and dibromopropamidine isethionate, is found to be effective but may be accompanied by drug toxicity and development of resistance [3].

Polyhexamethylene biguanide and chlorhexidine have proved to be effective with treatment success of 78% and 86% respectively [4]. However, side effects in the form of cataract, iris atrophy and peripheral ulcerative keratitis have been attributed to the use of those drugs [5].

Medicinal plants can be an alternative resource of novel anti-protozoal drugs with high effectiveness and low toxicity [6].

Nigella sativa seeds are considered one of the potential natural sources in folk medicine [7]. Crude extracts (aqueous and alcoholic) and essential oil of N. sativa proved to have many therapeutic effects. Nigella sativa alcoholic extract is found to be as effective as Metronidazole in the cure of giardiasis [8]. Aqueous extract has demonstrated a potential therapeutic effect against Blastocystis hominis [9] and Trichomonas vaginalis [10]. Nigella sativa oil was found to have a significant therapeutic effect on experimental infection with Cryptosporidium parvum [11], Trypanosoma brucei [12] and Toxoplasma gondii [13].

Wheat germ agglutinin (WGA) has produced dose-related growth inhibition of Giardia lamblia trophozoites in vitro [14].

So the aim of the present work is to study the in vitro effect of Nigella sativa aqueous and alcoholic extracts as well as wheat germ agglutinin on pathogenic Acanthamoeba.

Material and Methods

The current study was conducted at The Medical Parasitology Department, Faculty of Medicine, Ain Shams University during the period from June 2010 to June 2011.

Plant materials and extraction procedure:

Preparation of Nigella sativa aqueous extract [10]: Nigella sativa seeds, obtained from the local market, were washed to remove any debris and air-dried. A weight of 125 g seeds was crushed then boiled in distilled water (500 ml) for 90 minutes and filtered through gauze. The filtrate was evaporated under reduced pressure, lyophilized and preserved in the deep freezer (–20°C) till it was used.

Preparation of Nigella sativa alcoholic extract [15]: Nigella sativa seeds were freed of dust. A weight of 125 g seeds was crushed in a domestic grinder then soaked in absolute ethanol in a beaker for 5 days at room temperature. The amount of ethanol was just enough to adequately cover the seeds. They were filtered under UV light using a filter paper. Ethanol was evaporated from the filtrate using a rotary evaporator. The prepared extract was collected and stored at –20 °C.

Wheat germ agglutinin (WGA): It was obtained as a highly purified lyophilized powder L9640 (Sigma Corporation).

Preparation of the drug control: chlorhexidine 0.02% [4]: Chlorhexidine digluconate 20% was obtained from Sigma Corporation. Chlorhexidine 0.02% was prepared by diluting one (1) ul of chlorhexidine 20% with one (1) ml of distilled water. Nigella sativa aqueous and alcoholic extracts were mixed in distilled water to get concentrations of 500 μg/ml, 1 mg/ml, 5 mg/ml, 10 mg/ml, 15 mg/ml, 20 mg/ml, 25 mg/ml and 30 mg/ ml. Wheat germ agglutinin was dissolved in distilled water to get concentrations of 50, 100, 150, 250, 500 μg/ml, 1 mg/ml, and 2 mg/ml. Chlorhexidine was tested in a concentration of 0.02% as a drug control.

Acanthamoeba spp. Isolation

Corneal scrapings were obtained from patients attending The Corneal Outpatient Clinic, Research Institute of Ophthalmology, Giza, who were clinically suspected to have Acanthamoeba keratitis. The samples were kept in 5 ml screw-capped vials containing Page Amoeba Saline solution (PAS) to betransported to the laboratory for cultivation. The specimens were inoculated directly onto the surface of 1.5% non-nutrient agar (NNA) plates seeded with Escherichia coli bacterial suspension and incubated in a humidified chamber at 30°C [16]. The presence of Acanthamoeba could be seen by the clear tracks on the E. coli lawn NNA produced by the feeding trophozoites of Acanthamoeba. Examination of the agar plate surface for the presence of amoebic growth was carried out daily for up to 14 days with a light and inverted microscopes using x40 objective. Acanthamoeba was identified by the specific morphology of cyst and trophozoite. Subcultures were done after 2 weeks from positive cultures with confirmed amoebic growth by cutting a small square of agar using a sterile scalpel and placed upside down on new NNA-E.coli plates. The plates were incubated in humidified chambers at 30ºC and examined after 24 h. Performing sub-culturing several times facilitated the isolation of Acanthamoeba. Acanthamoeba cysts were collected from 3-week cultures. The agar surfaces were flooded with 5 ml of phosphate buffered saline (PBS) and were gently scraped with an inoculating loop. Cysts were harvested from the suspension by centrifugation at 350 × g for 10 min. The supernatant was aspirated, and the sediment was washed twice in PBS in order to eliminate most of the bacteria. Cysts in the resultant suspension were counted with a hemacytometer and the suspension was standardized to be 25×10⁴/ml [17].

Experimental design:

In order to evaluate the in vitro amoebicidal activity of Nigella sativa aqueous and alcoholic extracts as well as WGA. Chlorhexidine 0.02% as a drug control Acanthamoeba were incubated with different concentrations of Nigella sativa aqueous and alcoholic extracts (500 μg/ml and 1,5,10,15,20,25,30 mg/ml), and WGA (50,150,250,500 μg/ml and 1,2 mg/ml), for different incubation periods (24, 48 and 72 h). One hundred microliters (100 μl) of the calibrated trophozoite suspension (1×10⁵/ml) were inoculated into each well of a 96-well plate and then the plate was left for 30 min to avoid disturbance of the adherence of amoebae onto the wells’ surface. Then, the PBS solution was removed and 100 μl of each concentration of the plant extracts were added into wells. The plate was sealed and incubated at 30°C for different incubation periods. Also in a separate well 100 μl of the calibrated cyst suspension (25×10⁴/ml). In addition, controls containing only the parasite in PBS as a parasite control and parasite plus 0.02% chlorhexidine gluconate (prepared from a solution 20% in H2O CHX, C-9394; Sigma) as a reference drug control, were submitted to the same procedure. Each experiment was performed in triplicate. After each incubation period, 100 μl from each test and control wells were transferred into 100 μl of 0.3% basic methylene blue media. Unstained (viable) and stained (nonviable) parasite were enumerated in the haemocytometer, 10 min after stain addition. For cultures containing no viable cysts, an additional test was performed to confirm the results obtained. To evaluate their viability, it was inoculated onto NNA-E.coli plate, incubated at 30°C for additional 72 h and examined to detect any viable cysts or trophozoites [18].

Evaluation of the drug efficacy was done by:

• Counting the number of trophozoites ad cyst separetly using the haemocytometer after each period of incubation.

• Calculation of the percent of growth reduction according to the equation [19]

• Percent of growth reduction = a – b / a × 100

• Where; a = Mean number of trophozoites / cysts in parasite control cultures.

• b = Mean number of trophozoites / cysts in drug treated cultures.

• Determination of the minimal inhibitoryconcentration (MIC)as thelowest concentration ofthetestedplant extractsandchlorhexidine 0.02% in which no viable organism observed [20].

Statistical evaluations

Statistical analysis was performed using Microsoft Excel version 2010 and Statistical Package for Social Sciences (SPSS) for Windows version 15.0. Categorical variables were presented as number and percentage. The inhibition percentage caused by chlorhexidine 0.02% was taken as the reference values in different incubation periods (24, 48 and 72 hours). Differences between inhibition percentages of different concentrations of tested medications were compared to the reference drug using Yates’ corrected chi-squared test. Significance level was set at 0.05 while the highly significance level was set at 0.001.

Results

The results of the present study are shown in the Tables (1-6).

Table 1: Mean Number and Percentage of Inhibition of Viable Acanthamoeba Trophozoites Incubated with Nigella sativa Aqueous Extract for 24, 48 and 72 hrs.

Table 2: Mean Number and Percentage of Inhibition of Viable Acanthamoeba Cysts Incubated with Nigella sativa Aqueous Extract for 24, 48 and 72 hrs

Table 3: Mean Number and Percentage of Inhibition of Viable Acanthamoeba Trophozoites Incubated with Nigella sativa Alcoholic Extract for 24, 48 and 72 hrs.

Table 4: Mean Number and Percentage of Inhibition of Viable Acanthamoeba Cysts Incubated with Nigella sativa Alcoholic Extract for 24, 48 and 72 hrs.

Table 5: Mean Number and Percentage of Inhibition of Viable Acanthamoeba Trophozoites Incubated with WGA for 24, 48 and 72 hrs.

Table 6: Mean Number and Percentage of Inhibition of Viable Acanthamoeba Cysts Incubated with WGA for 24, 48 and 72 hrs.

Discussion

Acanthamoeba was first recognized as an ocular pathogen in 1973 and was the cause of an epidemic in the 1980s due to contaminated contact lenses [21].

A variety of drugs have been used for treatment of AK including chlorhexidine, polyhexamethylene biguanide, propamidine isethionate, dibromopropamidine isethionate, neomycin, paromomycin, polymyxin B, clotrimazole, ketoconazole, miconazole and itraconazole [22]. Miltefosine, a hexadecylphosphocholine, has also been shown to have amoebicidal potential [23].

Treatment failures occur and may be due to inherent or acquired resistance (perhaps attributed to strain and species susceptibility differences) or to advanced corneal disease [21]. The authors assumed that failures may also be due to the inability to achieve cysticidal concentrations of anti-amoeba agents in the cornea.

Interest in medicinal plants has burgeoned due to increased efficiency of new plant-derived drugs and the growing interest in natural products. Because of the concerns about the side effects of conventional medicine, the use of natural products as an alternative to conventional treatment in healing and treatment of various diseases has been on the rise in the last few decades. The use of plants as medicines dates from the earliest years of man's evolution [24]. Medicinal plants serve as therapeutic alternatives, safer choices or in some cases, as the only effective treatment. A larger number of these plants and their isolated constituents have shown beneficial therapeutic effects including anti-oxidant, anti-inflammatory, anti-cancer, anti-microbial and immuno-modulatory effects [25].

Among the promising medicinal plants is Nigella sativa, a dicotyledon of the Ranunculaceae family. It is an amazing herb with rich historical and religious background. It has been employed since long time as a spice and food preservative [7].

The black seed was referred to by the prophet Mohammed (PBUH)as having healing powers, he advised his followers “Hold on to use the Black cumin, for it has a remedy for every illness except death” [25]. Historically, it has been recorded that N. sativa seeds were prescribed by ancient Egyptians and Greek physicians to treat headache, nasal congestion, toothache and intestinal worms as well as a diuretic and increase milk production [26]. The seeds of N. sativa, known as black seed, black cumin or “Habatul-Barakah,” have long been used in folk medicine in the Middle and Far East as a traditional medicine for a wide range of illnesses including bronchial asthma, headache, dysentery, infections, obesity, back pain, hypertension and gastrointestinal problems [27-28].

The seeds of N. sativa are the source of the active ingredients of this plant [26]. By analysis of N. sativa oil, thymoquinone (TQ), dithymquinone (DTQ), thymohydroquinone (THQ) and thymol (THY) are considered the main active ingredients [29]. The seeds also contain other ingredients including nutritional components such as carbohydrates, fats, vitamins, mineral elements (calcium, iron and potassium) and proteins including eight of the nine essential amino acids [30]. They contain carotene which is converted by the liver to vitamin A [31].

Nigella sativa seeds were found to have phospholipids. The major separate individual phospholipid classes are phosphatidylcholine followed by phosphatidyle-thanolamine, phosphatidylserine and phosphatitdylinisitol, respectively [32]. The seeds also contain monosaccharides in the form of glucose, rhamnose, xylose and arabinose. They are rich in the unsaturated and essential fatty acids. The major unsaturated fatty acid is linoleic acid followed by oleic acid [33].

Lectins (agglutinins) are naturally occurring proteins recognized by their ability to bind, with high specificity, to glycosylated residues on parasite membrane. Many lectins are derived from common human foods such as beans and wheat [34]. No side effects were reported by human volunteers who were fed wheat germ agglutinin (WGA) at a large dose (200 mg) daily for 4 days [35]. Many trials were done to target carbohydrate residues on the surface membrane of protozoa by the use of lectins [14, 36]. A wide range of biological functions are mediated by lectin interaction with cell surface glycoproteins, including cellular differentiation and adherence [10].

In the present work, the in vitro effects of N. sativa aqueous and alcoholic extracts as well as WGA were studied in comparison to chlorhexidine (0.02%) as a drug control. Results of the present study can be considered as a preliminary report on the amoebicidal activity of those agents on Acanthamoebae.

Chlorhexidine (0.02%) showed a trophozoite inhibition percentage of 100% after incubation for 24, 48 and 72 hrs. However, it produced cyst inhibition percentages of 56%, 64% and 80% after the same durations respectively. Similar results were reported by [37] who stated that chlorhexidine had a great in vitro anti-acanthamoebal activity for several Acanthamoeba strains, both for cysts and trophozoites.

The MLC of N. sativa aqueous extract that had 100% inhibition percentage on trophozoites was 5 mg/ ml (after incubation for 24 hrs) and 500 μg /ml (after 48 hrs).

These results are nearly similar to those reported by several authors working on other protozoa. El Wakil [9] found that N. sativa aqueous extract (at concentrations of 100 and 500 μg /ml) had a potent lethal effect on two Blastocystis hominis isolates, but with different extent. The author also found that the concentration of 500 μg/ml had a significant inhibitory effect on the living cell rate of both isolates.

Also, the in vitro inhibitory activity of N. sativa aqueous extract was tested on the growth and motility of Trichomonas vaginalis in comparison to metronidazole [10]. The MLC was found to be 10 mg/ml after 48 hrs with a remarkable effect on T. vaginalis growth and motility.

Results of the present work showed that the MLC of N. sativa aqueous extract on Acanthamoeba cyst stage was 30 mg/ ml after 24 hrs incubation and 25 mg/ml after 48 hrs. These results show that higher concentrations are required to cause 100% inhibition of cysts compared to trophozoites. In addition, chlorhexidine (0.02%) caused inhibition percentages of 56%, 64% and 80% on Acanthamoeba cysts after incubation for 24, 48 and 72 hrs respectively. Likewise, [22] stated that Acanthamoeba trophozoites are sensitive to most available chemotherapeutic agents. Persistent infection is related to the presence of Acanthamoeba cysts, against which very few of these agents are effective. Also, the same finding was reported by Polat et al. [18] who stated that the sensitivity of Acanthamoeba trophozoites was higher than that of cysts.

Results of the present study reveal a more potent cysticidal activity of N. sativa aqueous extract on Acanthamoeba cysts compared to chlorhexidine (0.02%). The highest inhibition percentage achieved by chlorhexidine (0.02%) on Acanthamoeba cysts was 80% after 72 hrs incubation. The same result was obtained using N. sativa aqueous extract at a concentration of 10 mg/ml after the same duration. Moreover 100% inhibition was obtained by N. sativa aqueous extract after 48 hrs on increasing the concentration to 25 mg/ml or to 30 mg/ml after 24 hrs with a significant (p<0.05) and a highly significant (p<0.001) differences respectively.

The present work showed that the MLC of N. sativa alcoholic extract that gave 100% inhibition of Acanthamoeba trophozoites was 500 μg /ml (after incubation for 48 hrs) which was similar to that of the aqueous extract. After incubation for 24 hrs, the MLC was 10 mg/ml while that of N. sativa aqueous extract was 5 mg/ ml after the same duration.

Regarding the effect of N. sativa alcoholic extract on Acanthamoeba cysts, the MLC was 30 mg/ml after 24 hrs and 25 mg/ml after 72 hrs. These results are nearly similar to those obtained by N. sativa aqueous extract although 100% inhibition was achieved by 25 mg/ml concentration after 48 hrs incubation.

It is also evident that cysticidal activity of N. sativa alcoholic extract is more potent compared to chlorhexidine (0.02%).

The effect of N. sativa alcoholic extract on giardiasis was evaluated by Bishara and Masoud [9]. It was found to be as effective as metronidazole in the cure of giardiasis in experimental animals. Both gave a cure rate of 80% after a single oral dose of 40mg/kg body weight.

In a more recent study a strong anti-malarial activity of N. sativa ethanolic extract was reported by Abdulelah and Zainal-Abidin [38] against Plasmodium berghei in mice. Both intra-peritoneal and oral treatments showed suppressive activities (decreased parasitaemia and increased survival times of the infected mice) with the highest values noted by the 100 μL/ kg dose (70.59% & 86.19% suppression by intraperitonial and oral routes respectively).

The anti-acanthamoebal activity of N. sativa can be attributed to the various beneficial effects of the crude and purified components of its black seeds including antimicrobial effect. They also have mast cell stabilizing, anti-inflammatory as well as immune stimulatory activities [39,40]. Most of these biological activities of N. sativa have been attributed to thymoquinone, the main active constituent of the volatile oil extracted from the seeds [41]. Nigella sativa seeds contain phenolic anti-oxidants which are well known for their anti-inflammatory and antiparasitic properties [42]. The seeds also contain different classes of alkaloids that were believed to block protein synthesis of Plasmodium falciparum [43].

In the present work, the influence of WGA on pathogenic Acanthamoebae was studied in vitro.

The MLC of WGA which had an inhibition percentage of 100% on Acanthamoeba trophozoites was 50 μg /ml after 24 hrs incubation similar to chlorhexidine 0.02%.

Regarding the effect of WGA on Acanthamoeba cyst stage, concentration of 1 mg/ml caused more or less similar results to the drug control after all durations of incubation with statistically insignificant differences (p>0.05).

The anti-parasitic properties of WGA were investigated in several previous studies. Wheat germ agglutinin could inhibit Giardia lamblia excystation as effectively as monoclonal antibodies directed against cyst wall antigens. Excystation was induced in vitro by mimicking cyst passage through the stomach and upper small intestine. The in vitro-derived cysts were exposed to an acidic reducing environment followed by protease treatment at a slightly alkaline pH. Pre-exposure of cysts to polyclonal rabbit antiserum against purified cyst walls or to WGA (10ug/ml) inhibited excystation by >90% [36].

In the present study increasing the concentration of WGA to 2 mg/ ml resulted in inhibition of Acanthamoeba cysts at a percentage of 76% after 24 hrs incubation (compared to 56% by the drug control) and a significantly higher inhibition percentage of 80% after 48 hrs compared to 64% by the drug control (p<0.05). So it is expected that increasing the dose of WGA may result in complete inhibition of the more resistant Acanthamoeba cysts.

Then, the low concentration of 50 μg /ml was found lethal to Acanthamoeba trophozoites after 24 hrs incubation, while the relatively higher concentration of 2 mg/ml could not achieve 100% inhibition of Acanthamoeba cysts. Starting with such low concentrations of 50 μg/ml, 100 μg/ml, 150 μg/ml, 250μg/ml and 500 μg/ml in the present study goes in accordance with similar concentrations used effectively in previous studies against other protozoal infections.

Ortega-Barria et al. [14] reported that a concentration of 100 μg /ml of WGA inhibited the growth of Giardia trophozoites by 80% after 72 hrs incubation in vitro and decreased cyst passage in mice infected with the closely related Giardia muris. Also, applying the same dose (100μg/ml) for the same duration (72 hrs) resulted in 81.36% inhibition of Trichomonas vaginalis growth [10], while growth inhibition of 100% was produced at a concentration of 250μg/ml.

The in vitro inhibitory effect of WGA on Acanthamoeba may be explained on the assumption of some mechanisms suggested by Ortega-Barria et al. [14] for the in vitro inhibition of Giardia growth by WGA. They suggested that the lectin may agglutinate trophozoites preventing them from multiplying, may interfere with the function of surface glycoproteins or it may be cytotoxic to the parasites. Also, Mirhaghani and Warton [44] and Tonkal [10] suggested that growth inhibition may be attributed to the specificity of WGA for N-acetyl-D-glucosamine-containing residues in T. vaginal is membrane.

The effect of other natural products on Acanthamoeba growth was studied by other investigators. Ethanolic extract of propolis was found to have amoebicidal as well as cysticidal properties for Acanthamoeba trophozoites and cysts in a dose and a time dependant manner [45]. Also ethanol extracts of Arachis hypogaea L., Curcuma longa L. and Pancratium maritimum L. have cysticidal properties for Acanthamoeba castellanii [46].

In conclusion, preliminary evaluation of the anti-acanthamoebal activity of 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.

References

1. Visvesvara GS, Moura H, Schuster FL (2007) Pathogenic and opportunistic free-living amoebae: Acanthamoeba spp, Balamuthia mandrillaris, Naegleria fowleri, and Sappinia diploidea. FEMS Immunol Med Microbiol 50: 1-26.
2. Walochnik J, Duchene M, Eibl H, Aspöck H (2003) Treatment of Acanthamoeba keratitis: possibilities, problems, and new approaches. 3: 10-7.
3. Schuster FL, Visvesvara GS (2004a) Free-living amoebae as opportunistic and non-opportunistic pathogens of humans and animals. Inter J Parasitol34: 1001- 1027.
4. Lim N, Goh D, Bunce C, Xing W, Fraenkel G et al., (2008) Comparison of Polyhexamethylene Biguanide and Chlorhexidine as Monotherapy Agents in the Treatment of Acanthamoeba Keratitis. Am J Ophthalmol 145: 130- 135.
5. Dart JK, Saw V, Kilvington S (2009) Acanthamoeba keratitis: Diagnosis and treatment update. Am J Ophthalmol 148: 487- 499.
6. Freitas SF, Shinohara L, Sforcin JM, Guimaraes S (2006) In vitro effect of propolis on Giardia duodenalis trophozoites. Phytomed 13: 170-175.
7. Ali BH, Blunden G (2003) Pharmacological and toxicological properties of Nigella sativa. Phytother Res 17: 299- 305.
8. Bishara SA, Masoud SI(1992)Effect of Nigella sativa extract on experimental giardiasis. N Egypt J Med 7: 1-3.
9. El Wakil HS (2007) Evaluation of the in vitro effect of Nigella sativa aqueous extract on Blastocystis hominis isolates. JEgypt Soc Parasitol 37: 801-813.
10. Tonkal A (2009) In Vitro Antitrichomonal Effect of Nigella Sativa Aqueous Extract and Wheat Germ Agglutinin. JKAU Med Sci 16: 17-34.
11. El-Refaii MA (2003) Trials on therapeutic effect of Nigella sativa oil extract on Cryptosporidium parvum in experimentally infected mice. Egypt J Agricul Res 81: 805-837.
12. Ekanem JT, Yusuf OK (2008)Some biochemical and haematological effects of black seed (Nigella sativa) oil on Trypanosoma brucei-infected rats. Afr J Biotechnol 7: 153-157.
13. Rayan HZ, Wagih HM, Atwa MM (2011) Efficacy of Black Seed Oil from Nigella sativa against Murine Infection with Cysts of Me49 Strain of Toxoplasma gondii. PUJ 4: 165-176.
14. Ortega-Barria E, Ward HD, Keusch GT, Pereira ME (1994) Growth inhibition of the intestinal parasite Giardia lamblia by a dietary lectin is associated with arrest of the cell cycle. J Clin Invest 94: 2283-2288.
15. Hannan A, Saleem S, Chaudhary S, Barkaat M and Arshad MU (2008) Antibacterial activity of Nigella sativa against clinical isolates of methicillin resistant Staphylococcus aureus. J Ayub Med Coll  20: 72-74.
16. Init I, Lau YL, Arin FA, Foead AI, Neilson RS et al., (2010) Detection of free living amoebae, Acanthamoeba and Naegleria in swimming pools, Malaysia. Trop Biomed 27: 566–577.
17. Perrine K, Hermann BP, Meador KJ, et al.(1995) The Relationship of Neuropsychological Functioning to Quality of Life in Epilepsy. Arch Neurol 52: 997–1003.
18. Polat ZA, Vural A, Ozan F, Tepe B, Ozcelik S et al., (2008) In vitro evaluation of the amoebicidal activity of garlic (Allium sativum) extract on Acanthamoeba castellanii and its cytotoxic potential on corneal cells. J Ocul Pharmacol Ther 24: 8-14.
19. Palmas et al. (1984) Digestive Diseases and Sciences. 29:983-987.
20. Meingassner JG, Thurner J (1979) Strain of Trichomonas vaginalis resistant to metronidazole and other 5-nitroimidazoles. Antimicrob Agents Chemother 15: 254-257.
21. O'Day DM, Head WS (2000) Advances in the management of keratomycosis and Acanthamoeba keratitis. 19: 681-687.
22. Schuster FL, Visvesvara GS (2004b) Opportunistic amoebae: challenges in prophylaxis and treatment. 7: 41–51.
23. Walochnik J, Duchene M, Seifert K, Obwaller A, Hottkowitz T et al., (2002) Cytotoxic activities of alkylphosphocholines against clinical isolates of Acanthamoeba spp. Antimicrob Agents Chemother 46: 695–701.
24. Dattner AM (2003) From medical herbalism to phytotherapy in dermatology: back to the future. Dermatol Ther 16: 106– 113.
25. Salem ML (2005) Immunomodulatory and therapeutic properties of the Nigella sativa seeds. Int immunopharmacol 5: 1749-1770.
26. Goreja WG (2003) Black Seed: Nature’s Miracle Remedy. NewYork, Pp: 4-50.
27. Schleicher P, Saleh M (1998) Black seed cumin: the magical Egyptian herb for allergies, asthma, and immune disorders. Pp: 90.
28. Al-Rowais NA (2002) Herbal medicine in the treatment of diabetes mellitus. Saudi Med J 23: 1327- 1331.
29. Ghosheh OA, Houdi AA, Crooks PA (1999) High performance liquid chromatographic analysis of the pharmacologically active quinones and related compounds in the oil of the black seed (Nigella sativa). J Pharm Biomed Anal 19: 757– 762.
30. Chun H, Shin DH, Hong BS, Cho WD, Cho HY et al., (2002) Biochemical properties of polysaccharides from black pepper. Biol Pharm Bull 25: 1203– 1208.
31. Al-Jassir MS (1992) Chemical composition and microflora of black cumin (Nigella sativa) seeds growing in Saudi Arabia. Food Chem 45: 239– 242.
32. El-Mahmoudy A, Matsuyama H, Borgan MA, Shimizu Y, El-Sayed MGet al., (2002): Thymoquinone suppresses expression of inducible nitric oxide synthase in rat macrophages. Int Immunopharmacol 2: 1603– 1611.
33. Nickavar B, Mojab F, Javidnia K, Amoli MA (2003) Chemical composition of the fixed and volatile oils of Nigella sativa from Iran. Z Naturforsch C 58: 629–631.
34. Nachbar MS, Oppenheim JD (1980) Lectins in US diet: Survey of lectins in commonly consumed foods and a review of literature. Am J Clin Nutr 33: 2338-2345.
35. Lorenzsonn V and Olsen WA (1982) Invivo responses of rat intestinal epithelium to intraluminal dietary lectins. Gastro-enterol. 82: 838-848.
36. Meng TC, Hetsko ML, Gillin FD (1996) Inhibition of Giardia lamblia excystation by antibodies against cyst walls and by wheat germ agglutinin. Infect Immun 64: 2151-2157.
37. Narasimhan S., Madhavan HN and Therese L (2002) Development and application of an in vitro susceptibility test for Acanthamoeba species isolated from keratitis to polyhexamethylene biguanide and chlorhexidine. Cornea 21: 203–205.
38. Abdullah HA, Zainal-Abidin BA (2007) In vivo anti-malarial tests of Nigella sativa (black seed) different extracts. Am JPharmacol Toxicol 2: 46-50.
39. Mutabagani A, El-Mahdy SA (1997)Study of the anti-inflammatory activity of Nigella sativa and thymoquinone in rats. Saudi Pharm J 5: 110–113.
40. Swamy SM, Tan BK (2000) Cytotoxic and immunopotentiating effects of ethanolic extract of Nigella sativa seeds. JEthnopharmacol 70: 1– 7.
41. Elias JA, Lee CG, Zheng T, Ma B, Homer R et al., (2003) New insights into the pathogenesis of asthma. J Clin Invest 111: 291–297.
42. Ma Q, Kinner K (2002) Chemoprevention by phenolic antioxidants. J Biol Chem 277: 2477- 2484.
43. Morikawa T, Xu F, Ninomiya K, Matsuda H, Yoshikawa M (2004) Nigellamines A3, A4, A5, and C, new dolabellane-type diterpene alkaloids, with lipid metabolism-promoting activities from the Egyptian medicinal food black cumin. Chem Pharm Bull 5: 494–497.
44. Mirhaghani A, Warton A (1998) Involvement of Trichomonas vaginalis surface-associated glycoconjugates in the parasite/target cell interaction. A quantitative electron microscopy study. Parasitol. Res 84: 374–381.
45. Topalkara, Aysen, Vural Ayse, Polat, Zubeyde, et al., (2007) In Vitro Amoebicidal Activity of Propolis on Acanthamoeba castellanii. J ocul pharmacol herap 23 : 40-5.
46. El-Sayed NM, Ismail KA, Ahmed SA, Hetta MH (2011) In vitro amoebicidal activity of ethanol extracts of Arachishypogaea L, Curcuma longa L. and Pancratiummaritimum L. on Acanthamoebacastellanii cysts. Parasitol Res 110: 1985-1992.