Comparison Between the Effect of Metronidazole and some Medical Herbs Extracts on Viability of Entamoeba histolytica Trophozoites in Vitro

Amoebiasis is is widely distributed in Iraq. In order to search for alternative new antiamebic drugs, a study was planned to evaluate the efficacy of metronidazole and some medical herbs on the viability of Entamoeba histolytica trophozoites in vitro culture. E. histolytica was isolated from stool examination of patients attended Pediatric Hospital Kirkuk, using wet mount technique. The active trophozoites were cultured in Lock egg medium. After isolation of trophozoites tested against various concentrations of metronidazole, and medical herbs including Artemisia herba alba, Punica granatum and Tecurium polium , using viability Pandroff tube, which contain Jones broth medium. Both aqueous and alcoholic extractions of medical herbs were used, at concentrations of 50%, 25%, 12.5% and 6.25%. Results: Metronidazole had highest inhibitory effect on viability of E. histolytica trophozoites than medical herbs. Medical herbs, Artemisia herba alba, recorded highest inhibitory properties against Entamoeba histolytica, followed by Punicaa granatum and Tecurium polium. The effect of alcoholic extract of all medical herbs was greater than aqueous extracts Kirkuk University Journal /Scientific Studies (KUJSS) Volume 11, Issue 3, September 2016 , p.p(278-289) ISSN 1992 – 0849 Web Site: www.kujss.com Email: kirkukjoursci@yahoo.com, kirkukjoursci@gmail.com 872 Conclusions: The efficacy of metronidazole was greater than medical herbs. The alcoholic extraction of medical herbs had greatest effect on viability of trophozoites than aqueous ones. Medical herbs can be used as an alternative of metronidazole.


INTRODUCTION
Eradication of Entamoeba histolytica may be difficult. Available treatments using metronidazole, diloxanide furate, tetracycline and others are imperfect at best and have toxic side effect such as gastric upset, optic atrophy, bitter taste, dermatitis and possibly carcinogenic [1]. In addition, treatment failures in as many as 20% of cases raise concern about possible resistance to the used drugs and especially during pregnancy [2].
Metronidazole is the drug of choice for amoebiasis although failure following metronidazole therapy has been reported in some cases of liver abscess due to E. histolytica [3].
Medical herbs have recently been widely used around the world due to their natural constituent and absence of any side effect. Medical herbs used were selected for their efficiency and prosperities in treatment of abdominal disturbance, amoebic dysentery, and other intestinal parasitic and bacterial infection [4].
This study was planned to evaluate the amoebic potency of metronidazole and some medical plant extracts (Artemisia herba-alba, Punica granatum and Tecurium polium).

Collection of stool samples
Stool samples were collected from Kirkuk Pediatric hospital, Kirkuk General Hospital, Primary Health Care Center (PHC) from beginning of February 2014 to end of January 2015.
The samples were examined for the presence of Entamoeba histolytica using wet mount technique. When the samples showed active trophozoite motility they were cultured in Boeck and Dboholve's Locke egg medium [5].  [6].

Preparation of Medical herbs
Plants were obtained from a local grocery which patronizes traditional plants in Kirkuk province, and the species were confirmed in the traditional plant center/ College of Science / Tikrit University. Medical herbs were firstly cleaned from dust, unwanted parts excluded using sieve. They were cut thoroughly into small parts to increase surface area; the 500 mg of plant was weighted. Added to Pyrex beaker, 500 ml of Distilled Water was added into beaker.
The mixture was boiled for one hour by a hot plate at 60˚C. Heat source was removed and the beaker left to cool at room temperature. Solution filtered by sterile gauze then by filter paper.
(Schleichers and Schuell GMbh filter paper, Germany). The filtrate was transferred into a suitable plastic container, and kept in refrigerator until used.
Two methods were used in medical plants preparation; Maceration method, which is also called alcoholic extraction, the extraction solvent was ethanol 95% [7] and Infusion method.
in which, the lighter parts of plants (leaves, flower, light stems) were steeped in boiled water for 15-30 minute [8].

Results
Table (1) show the effect of three concentration of metronidazole on Entamoeba histolytica during days of incubation, totally reduction of viability at (100 mg /ml) was occur on day 3; on day 6 at 50 mg/ml and 25 mg/ml. Artemisia herba Alba aqueous extraction shows good lethal activity, especially at concentration 50% when totally reductions occur on day 8; and on day 10 at concentration 25%, 12.5% and 6.25%, with significant difference (P<0.05) in comparison to the control.
Alcoholic extraction of Artemisia herba-alba shows high efficacy in killing parasite in comparison to control (P<o.o5), totally reduction of viability occurred on day 6 at concentration 50% and 25%, and on day 8 at 12.5% and 6.25% concentration Table (2). Aqueous extraction of Tecurium polium shows low effect on viability of Entamoeba histolytica reduction of viability occur on day 10 in concentrations 50% , 25% and 12.5%, on day 12 at concentration 6.25%, Tecurium polium showed non significant effect at concentration 12.5% and 6.25%. The viability of Entamoeba histolytica treated with alcoholic extraction of Tecurium polium was affected very slowly. Total reduction of viability occur on day 10 at 50%, 25%, and 12.5%, and on day 12 at concentration 6.25% but the results were considered significant statistically (P<0.05) as seen in Table (4).

Discussion
Metronidazole treated tubes showed high declining in trophozoite count, total reducing of viability reached at 3 rd day in 100 mg/ ml concentration, and on 5 th day and 6 th day in 50mg /ml and 25 mg/ ml respectively. In a relative study metronidazole showed total reducing of viability after 48 hours at 100 g/ ml [9]. Metronidazole activity was due to trapping electrons by virtue of its very low redox potential. The generation of H2 from pyruvate is halted and the organism soon becomes depleted of NADH and NADPH. The nitro ring of metronidazole becomes cleaved in the process producing toxic substances that hasten cell death [10].
Metronidazole requires at least 10 days at high dosage to eradicate luminal amoebae [11].
Aqueous extraction of Artemisia herba-Alba shows significant effect on reduction of the viability of Entamoeba histolytica, complete reduction of viability occurs on day eight of incubation using 50% concentration and on day nine in 25%, 12.5%, 6.25% concentrations.
The efficacy of alcoholic extraction was greater than the aqueous one, total reduction of viability reported after four days of incubation in 50% and 25% concentration, and after seven days in 12.5% and 6.25% concentration. From these results, it appears that alcoholic extraction of Artemisia herba alba was more effective than the aqueous ones in the inhibition of the Entamoeba histolytica growth and considers very close to effect of metronidazole. AntiParasitic effect of Artemisia herba alba may be due to the presence of sesquiterpene lactones [12], and flavonoids [13], thujone, santonin, the sterols beta-sitosterol and stigmasterol [14].
The same results for Punica granatum aqueous and alcoholic extraction effect were reported against Entamoeba histolytica, except variance in the trophozoite count which is lower in alcoholic extraction. Total reduction of viability occur on day 7 from incubation at 50% and 25% concentration and occur in day 8 and 9 at 12.5% and 6.25% respectively. In relative studies (15) showed that alcoholic extraction for Punica granatum was more effective than aqueous ones in inhibitory properties. infection in which its action was attributed to alkaloids [17]. Although Punica granatum contain pelleltierine alkaloids which are considered as antihelminthic (especially cestodes) and large scale of protozoa [18], several studies showed that extraction of Punica granatum roots, rinds, and bark can be treated as amoebic dysentery because these components change the protinous nature of the intestine and reduces fluid flow, in addition to their antibacterial and relieving of bacterial poisons [19].
Aqueous extraction of Tecurium polium shows low effect on the viability of Entamoeba histolytica, which persists until day 10 at 50% and 25% concentrations, while no significant differences were recorded in comparison to control at 12.5% and 6.25%, alcoholic extraction shows results near to that recorded from aqueous extraction except that it shows significant differences at 12.5% and 6.25% concentration in comparison to control. The herbs possess hypoglycemic, insulintrobic, and anti-inflammatory activities [20]. Tecurium polium have antispasmodic and anti bacterial activity [21]. These properties promote the use of Tecurium against Entamoeba histolytica may be due to antioxidant prosperities which promote the anaerobic condition [22], and the anaerobic conditions are preferred by Entamoeba histolytica to grow at good scale in the culture medium [23].
It is concluded that the effect of metronidazole is the best against E. histolytica in vitro studies in comparison with medical herbs. Medical herbs (Artemesia herba alba and Punica granatum) have competent effect on E. histolytica viability, while Tecurium polium had lowest effect on viability of trophozoite. The effect of alcoholic extracts was greater than aqueous ones. TLC could be used for isolation and identification of chemical compounds. The best solvent system was the solvent consisting of butanole, acetic acid and distilled water.
It is recommended to carry on in vivo studies in order to determine the best concentration that can be used against amebic dysentery. Specific studies on medical herbs should be done with more advanced techniques like HPLC in order to identify the active chemical compounds present in medical herbs and to use it as alternative for chemical drugs with undesired side effects.