Antimicrobial Effects of Thymoquinone on Entamoeba histolytica and Giardia lamblia

Protozoa are one of the three chief types of parasites that lead to diseases in humans. Protozoan Infections are contagious and can be transmitted via the fecal-oral route.1,2 Blood protozoa can be transmitted through a third source such as insects.3 There are four main groups of protozoa according to how they move. That includes the sarcodina (ameba), mastigophora (flagellates), ciliophora (ciliates) and the Sporozoa.4,5 Many parasitic diseases are known to clinicians and public health professionals, which includes trichomoniasis, babesiosis, malaria, toxoplasmosis, leishmaniasis, african sleeping sickness, giardiasis and amoebiasis.6 Giardiasis is transmitted through oral contact and caused by Giardia lamblia and known as intestinal protozoan diseases.7 Amoebiasis is caused by sarcodina group of protozoa (Such as Entamoeba histolytica) that invade body tissues.8,9 The ailments triggered by protozoan parasite are accountable for substantial mortality and morbidity, distressing more than 500 million of the world population. The epidemiological management of protozoan is insufficient due to complicatedness of vector and reservoir control; while the progress in the development of protozoan vaccine is slow and arduous. Currently, the chemotherapy stays necessary constituent of both clinical management and disease control programmer in prevalent areas. The medications in utilization as anti-protozoan agents were discovered over 60 years and a number of issues limit their usefulness such as: high cost, drug resistance, poor compliance, low effectiveness and deprived safety. Recently, the searches for new drugs against protozoa parasite have been increased and more interest has been put on the traditional medicine, especially herbal remedies.10,11 Seeds’ oil of Nigella sativa (Ranunculaceae), is widely used as a food and cure.12,13 Experimental studies confirmed that the plant is respiratory stimulant, diuretic, hypoglycemic, anti-inflammatory, antioxidant, anticancer, antimicrobial and analgesic. Previous phytochemical reports showed that the seed contain alkaloids, tannins, steroids and flavonoids.13-15 Thymoquinone (TQ), an active ingredient of Nigella sativa, has been reported to exhibit anti-oxidant, anti-inflammatory, antimcrobial and anti-tumor activities. The current study was designed to assess the Effects of thymoquinone on Entamoeba histolytica and Giardia lamblia.


INTRODUCTION
Protozoa are one of the three chief types of parasites that lead to diseases in humans.Protozoan Infections are contagious and can be transmitted via the fecal-oral route. 1,2Blood protozoa can be transmitted through a third source such as insects. 3There are four main groups of protozoa according to how they move.That includes the sarcodina (ameba), mastigophora (flagellates), ciliophora (ciliates) and the Sporozoa. 4,5Many parasitic diseases are known to clinicians and public health professionals, which includes trichomoniasis, babesiosis, malaria, toxoplasmosis, leishmaniasis, african sleeping sickness, giardiasis and amoebiasis. 6Giardiasis is transmitted through oral contact and caused by Giardia lamblia and known as intestinal protozoan diseases. 7Amoebiasis is caused by sarcodina group of protozoa (Such as Entamoeba histolytica) that invade body tissues. 8,9he ailments triggered by protozoan parasite are accountable for substantial mortality and morbidity, distressing more than 500 million of the world population.The epidemiological management of protozoan is insufficient due to complicatedness of vector and reservoir control; while the progress in the development of protozoan vaccine is slow and arduous.Currently, the chemotherapy stays necessary constituent of both clinical management and disease control programmer in prevalent areas.The medications in utilization as anti-protozoan agents were discovered over 60 years and a number of issues limit their usefulness such as: high cost, drug resistance, poor compliance, low effectiveness and deprived safety.Recently, the searches for new drugs against protozoa parasite have been increased and more interest has been put on the traditional medicine, especially herbal remedies. 10,11eeds' oil of Nigella sativa (Ranunculaceae), is widely used as a food and cure. 12,13Experimental studies confirmed that the plant is respiratory stimulant, diuretic, hypoglycemic, anti-inflammatory, antioxidant, anticancer, antimicrobial and analgesic.4][15] Thymoquinone (TQ), an active ingredient of Nigella sativa, has been reported to exhibit anti-oxidant, anti-inflammatory, antimcrobial and anti-tumor activities.The current study was designed to assess the Effects of thymoquinone on Entamoeba histolytica and Giardia lamblia.

Parasite isolate
Entamoeba histolytica and Gardia lambelia used in all experiments were taken from patient.All positive samples were examined by wet mount preparation.Trophozoites of the two parasites were performed at 37 ± 1°C in RPMI 1640 medium containing 5% of bovine serum.The trophozoites were maintained for the assays and were employed in the log phase of growth.Parasites were counted under the microscope by haemocytometer chamber.

In vitro susceptibility assays
In vitro susceptibility assays of the current research used the sub-culture method. 16This is highly stringent and sensitive method for assessing the anti-protozoal effects (gold standard) particularly in Entamoba histolytica, Gairdia intestinalis and T. vaginalis.Five mg from the compound was dissolved in 5% dimethyl sulfoxzide (5000 ppm).The concentrates were stored at -20°C for further analysis.Sterile 96-well microtite plate was used for test material, positive control and negative control.The final volume in the wells was 100 μl.Each test included metronidazole pure compound [(1-(2-hydroxyethl)-2-methyl-5 nitroimidazole], a drug was used as positive control in concentration 312.5 µg/µml, whereas untreated cells were used as a negative controls (culture medium plus trophozoites).Samples were taken for counting at 0, 24, 48, 72, 96, and 120 hours.For counting, the samples 169 were mixed with Trypan blue in equal volumes The final number of parasites was determined with haemocytometer in triplicate.The mortality % of parasite for each extract activity was carried out according to the following formula:

Statistical analysis
All data were presented as means ± S.D. Statistical analysis for all the assays results were done using Microsoft excel program.Student t test was used to determine significant difference between control and plant extracts at level of P<0.05.

RESULTS AND DISCUSSION
Medicinal chemists have discovered an amount of medications which can be utilized against numerous but by far not all endoparasites.A main difficulty is that many of these drugs were synthesized many years ago and some parasites have become resistant to them.The development of novel antiparasitic medications has not been much of precedence for the pharmaceutical industry because many of the parasitic diseases happen in deprived countries where the populations cannot give to pay a elevated price for the drugs.Thus an investment in drug progress against parasitic diseases is a hazardous matter.8][19] The current study was designed to assess the Effects of thymoquinone on Entamoeba histolytica and Giardia lamblia.Seeds' oil of Nigella sativa (Ranunculaceae), is widely used as a food and cure. 12,13Experimental studies confirmed that the plant is respiratory stimulant, diuretic, hypoglycemic, anti-inflammatory, antioxidant, anticancer, antimicrobial and analgesic.4][15] Thymoquinone (TQ), an active ingredient of Nigella sativa, has been reported to exhibit anti-oxidant, anti-inflammatory, antimcrobial and anti-tumor activities.Thymoquinone was tested on both Entamoeba histolytica and Giardia lamblia using the range of concentrations 0-200 µg/mL.Figure 1 indicates that the mortality of TQ showed 85.5%, 91.5% and 96.8% mortality on E. histolytica for 25 ppm at 24 hr, 48 and 72 hr, respectively, with IC 50 2×10 -19 ,.On the other hand, this natural compound showed a mortality of 82.83%, 91.76% and 96.62% mortality on G. lamblia for 25 ppm at 24 hr, 48 and 72 hr, respectively, with IC 50 4.8×10 -5 as shown in Figure 2. The compound was also tested for 48 and 72 hours on both parasites.
The current results indicate that TQ is more potent on E. histolytica compared to G. lamblia.Metrondizole powder gave 70.9% mortality at 156 ppm at the same times.N. sativa seeds were found to turn render the parasite susceptible to damage by the host and may play a role in the anti-schistosomal potency.1][22] Dietary intake of chloroquine with N. sativa was examined previously and found effective in malaria's mice model of plasmodium berghei. 22,23. sativa seed in ethnomedicine and in modern years for the cure of microbial diseases has been used without any known side-effects.Consequently, N. sativa can offer a precious medication for microbial ailments. 24However, supplementary research is needed to assess and discover the specific cellular and biomolecular mechanisms of the antimicrobial effects of TQ, only or in mixture with other drugs.

Figure 1 :
Figure 1: In vitro activity of Thymoquinone against Giardia lambelia; mortality percentage in relation to time

Figure 2 :
Figure 2: In vitro activity of Thymoquinone against Entamoeba histolytica; mortality percentage in relation to time