Parasitol Res (2012) 111:5964 DOI 10.

1007/s00436-011-2801-x

ORIGINAL PAPER

Amoebicidal activity of the rhizomes and aerial parts of Allium sivasicum on Entamoeba histolytica
S. Degerli & S. Berk & B. Tepe & E. Malatyali

Received: 12 December 2011 / Accepted: 19 December 2011 / Published online: 4 January 2012 # Springer-Verlag 2011

Abstract Amebiasis is a severe illness caused by Entamoebachistolytica. The aim of this study is to evaluate the in vitro amebicidal activity of the rhizomes and aerial parts of Allium sivasicum, an endemic plant species from the flora of Turkey. Both extracts showed a time- and dose-dependent amebicidal action on the trophozoites. Among the extracts tested, rhizomes of A. sivasicum showed the strongest amebicidal effect on the trophozoites. In the presence of the rhizome extract at 2.0 mg/ml concentration, all of the trophozoites available in media have completely been killed within the 72nd hour. At 4.0 mg/ml extract concentration, all of the trophozoites available in media have completely been killed by the rhizome extract from the time of 24th hour. At 32.0 mg/ml extract concentration, 73.7% of the trophozoites were successfully killed by the extract within the first experimental hour. Aerial part extract at 4.0 mg/ml concentration completely killed the trophozoited within the 48th hour of the experimental procedure. At 8.0 mg/ml extract concentration, all of the trophozoites available in media have completely been killed by the aerial part extract from the time of 24th hour. At 32.0 mg/ml extract concentration, 67.7% of the trophozoites were successfully killed by the extract within the first experimental hour. These results suggest that the plant species evaluated here is a potential therapeutic drug for the treatment of Entamoeba
S. Degerli : E. Malatyali Department of Parasitology, School of Medicine, Cumhuriyet University, 58140 Sivas, Turkey S. Berk : B. Tepe (*) Department of Molecular Biology and Genetics, Faculty of Science and Literature, Cumhuriyet University, 58140 Sivas, Turkey e-mail: bektastepe@yahoo.com

infections, but it still needs to be evaluated quantitatively for determining the active phytochemicals.

Introduction Amebiasis is an infectious diseases caused by Entamoeba histolytica. It results in severe liver and brain abscess. It also causes high rate of morbidity and mortality in humans. In the treatment of amebiasis, for several decades, metronidazole, which is also known as 5-nitroimidazole, has been the drug of choice (Lichtenstein et al. 2005). On the other hand, there are some concerns about the use of this drug. These concerns have mainly been focused on its carcinogenicity (Upcroft and Upcroft 2001). Additionally, recent studies have reported several toxic effects such as genotoxicity, gastric mucus irritation, and spermatozoid damage (el-Nahas and el-Ashmawy 2004; Purohit and Basu 2000). According to some reports, failure with metronidazole could probably be heralding the development of drug resistance clinically (Gomez et al. 1996). Even after treatment with metronidazole, recurrence of amebic liver abscess has been reported and parasites may survive in spite of adequate treatment (Pittman and Pittman 1974). The genus Allium is one of the major sources of dietary flavonoids, which are a group of polyphenolics, in many countries (Hertog et al. 1995; Knekt et al. 1996). The genus Allium is very large consisting of more than 700 species of bulbous perennials and biennials that occur in temperate regions of the northern hemisphere (Knemann 1999). There are 164 Allium species available in Turkish flora, and 65 of them are endemic of which one of them is Allium sivasicum (Figs. 1 and 2; Davis 1984, 1998; Guner et al. 2000). Since antiquity Allium plants have been believed to be beneficial to human health. Garlic, a member of this

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Materials and methods Preparation of the methanol extracts The air-dried and finely ground samples were extracted by using a method described elsewhere (Sokmen et al. 1999). Briefly, the sample, weighing about 100 g, was extracted in a Soxhlet apparatus with methanol at 60C for 6 h. Due to the aqueous characteristics of the experimental media, mainly the water-soluble parts of the extracts could be used only. For obtaining the aqueous subfraction of the extracts, it was further fractionated with chloroform and distilled water. Finally, the extracts were then lyophilized and kept in the dark at +4C until tested. Extract yields of the polar subfractions of the rhizome and aerial part samples were determined as 3.26% and 4.17% w/w, respectively. Trophozoites E. histolytica strain was kindly provided by Dr. John Clark (London School of Hygiene & Tropical Medicine). It was a pathogenic strain isolated from a Bangladeshi child with acute amebic dysentery. Trophozoite forms of parasite were cultured monoxenically in the Robinson medium at 35.5C (Robinson 1968). Agar slants were prepared in screw cap glass bijou bottles; 1.5 ml of phthalate, 1.5 ml of BRS medium, 50 l of erythromycin, and 100 l Bacto peptone were mixed in the bottle, and sterilized rice starch is added to the medium before inoculation. Each time 0.5 ml of culture is used for inoculation and culture was renewed for a further 96 h to maintain the amoeba strain. The trophozoites in the log phase of growth were harvested by centrifugation at 500g at room temperature for 5 min. The pelleted cells were resuspended in phosphate buffer saline, the final concentration was adjusted to 10104 trophozoites/ ml, and the trophozoites were used in the assay without delay. The number of viable trophozoites was determined by trypan blue exclusion and direct trophozoite counts on a hemocytometer prior to experiment. Determination the amebicidal activity of plant extracts Eppendorf 1.5-ml microcentrifuge tubes with lids were used in the present study. The plant extracts were dissolved and diluted in sterile distilled water to serial concentrations (2, 4, 8, 16, 32, and 64 mg/ml). Two hundred fifty microliters of the calibrated trophozoite suspension and the same volume of test solution were mixed thoroughly via pipetting up and down. Then the tubes were kept at 35.5C in standard bacteriological incubator (Electro-Mag) for 1, 2, 3, 6, 8, 24, 48, and 72 h. The same procedure was applied to controls containing only trophozoite suspension and sterile

Fig. 1 A. sivasicum (figure has been obtained from www.plantbuzz.com)

genus, has held a special position as a prophylactic and therapeutic agent in cultures of Egypt, India, Greece, and Rome, a position it still holds in present-day societies. Anectodal evidence supports the important roles of the members of this genus in the prevention and treatment of pathogenic infections, tumors, and cardiovascular diseases (Cao et al. 1996; Gazzani et al. 1998; Yin and Cheng 1998). Additionally, they are thought to have also anticoagulant, anticancer, and antiparasitic effects, which are probably due to the specific organosulfur compounds such as ajoene, alliin, and allicin (Dorant et al. 1993; Mansell and Reckless 1991; Lun et al. 1994; Block 1985; Sendl et al. 1992). Our research is mainly focused on plant extracts for the treatment of parasitic infections effectively as an alternative way to the classical chemical and/or synthetic methods. In this context, amebicidal activities of some plant species have been reported by our research team (Tepe et al. 2011, 2012; Goze et al. 2009; Akin Polat et al. 2007a, b, 2008; Malatyali et al. 2012a, b; Degerli et al. 2012). Drugs of natural origin have already been used to treat other parasitic diseases (Arrieta et al. 2001; Kayser et al. 2003; Said Fernndez et al. 2005). The aim of this study is to evaluate the in vitro amebicidal activity of the water-soluble subfraction of methanolic extract obtained from A. sivasicum rhizomes and aerial parts. As far as our literature survey could ascertain, no report is available for the amebicidal activities of A. sivasicum against E. histolytica in the literature. Therefore, this study could be assumed as the first report on this topic.

Fig. 2 Distribution of Allium sivasicum (figure has been obtained from Turkish Plants Data Service, http://wwweski.tubitak.gov.tr/tubives/)

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distilled water. Cell growth was periodically monitored by light microscopy (Nikon, Eclipse E 200). Trypan blue dye exclusion test was used to determine the effect of extracts on the proliferation of parasite. Following the incubation periods at 35.5C, 25-l parasite suspensions were simply mixed with the same volume of trypan blue (0.5 mg/ml) in counting chamber. The mixture was allowed to incubate 23 min at room temperature, and then the unstained (viable) and stained (nonviable) cells were counted separately. Approximately a hundred of E. histolytica trophozoites were examined in each time, and all the tests were repeated three times. Statistical analysis The data were presented as mean values with standard deviations and analyzed by repeated measures of ANOVA followed by Tukey test for post hoc pairwise comparisons. The P value was set at 0.05 for significance level.

Results and discussion Amebiasis is a severe illness caused by E. histolytica. It is a kind of protozoan parasite 10 to 60 m in length. It moves through the extension of finger-like pseudopods (John and Petri 2006). Spread occurs via the fecaloral way, usually by poor hygiene during food preparation processes and eating or by the use of night soil (crop fertilization with human waste), as well as by oralanal sexual habits. Crowding and poor hygiene contribute to its prevalence in the continents of Asia, Africa, and Latin America. Fifty million people suffer from amebic colitis and liver abscess caused by E. histolytica resulting in 50,000 to 100,000 deaths yearly (Reed 2001; Tanyuksel and Petri 2003). The stable reservoir of infective cases causes difficulties in eradication. After malaria, it is likely that E. histolytica is the worlds second leading protozoan cause of death (Petri and Singh 1999). Among children under 5 years who were admitted with acute diarrhea in a hospital, E. histolytica was confirmed
Table 1 Effect of A. sivasicum rhizomes on the proliferation of E. histolytica trophozoites

in 7.8% of the cases (Suwatana 1997). The estimated number of infected cases may be much higher due to asymptomatic diseases and lack of a sensitive and specific diagnostic test (Petri et al. 2000; Behnia et al. 2008). The most effective and commonly used drug for treatment of intestinal protozoan infection is metronidazole. However, this drug has been reported to have unpleasant side effects such as metallic taste, headache, dry mouth, and to a lesser extent nausea, glossitis, urticaria, pruritus, and dark-colored urine. In addition, carcinogenic, teratogenic, and embryogenic effects have been documented (Upcroft et al. 1999; Upcroft and Upcroft 2001). Thus, the search for alternative antiamebic compounds with high activity, low toxicity, cheaper, and more effective is still a necessary goal. Plants are known to synthesize secondary metabolites that protect them from pathogens, insects, and mammalian herbivores (Tan and Zou 2001). They are potential sources of novel bioactive natural products for medicinal or agricultural applications. Extracts of medicinal plants including Ipomoea sp., Kaempferia galanga, and Cananga odorata showed acanthamebicidal activities (Chu et al. 1998). Several other drugs exhibited amebicidal or amebistatic effects against Acanthamoeba trophozoites and cysts in vitro. These include antibiotics as well as antifungal, antiviral, antiprotozoal, antipsychotic, and anticancer agents (Mattana et al. 2004; Osato et al. 1991; Schuster and Visvesvara 1998; Walochnik et al. 2002). Amebicidal activities of some plant species have previously been reported by our research team (Tepe et al. 2011, 2012; Goze et al. 2009; Akin Polat et al. 2007a, b, 2008; Malatyali et al. 2012a, b; Degerli et al. 2012). In vitro amebicidal effects of polar subfractions obtained from the methanol extracts of A. sivasicum rhizomes and aerial parts are presented in Tables 1 and 2, respectively. In the presence of extracts (ranging from 1.0 to 32.0 mg/ml), numbers of the viable E. histolytica trophozoites were decreased during the experimental process (from 1st to 72nd hours). Both extracts showed a time- and dosedependent amebicidal action on the trophozoites. Among

Dose (mg/ml)

Experimental periods 1h 3h 17.32.1 32.30.6 44.01.7 55.30.6 63.31.5 75.01.0 91.31.2 6h 8.71.2 12.31.5 21.71.5 30.31.5 49.32.3 64.01.7 90.71.2 8h 0 0 0 12.30.6 15.70.6 32.32.5 89.30.6 24 h 0 0 0 0 10.70.6 14.01.7 89.01.7 48 h 0 0 0 0 5.30.6 9.31.2 88.71.5 72 h 0 0 0 0 0 6.71.2 87.30.6

32.0 16.0 8.0 4.0 2.0 1.0 Control

26.31.5 43.71.5 59.33.1 71.72.1 83.31.5 91.70.6 93.01.7

Data were expressed as meanSD

62 Table 2 Effect of the aerial parts of A. sivasicum on the proliferation of E. histolytica trophozoites

Parasitol Res (2012) 111:5964

Dose (mg/ml)

Experimental periods 1h 3h 19.71.5 35.71.2 47.72.1 57.02.0 68.32.9 78.32.1 92.31.5 6h 13.01.7 15.71.2 24.31.2 31.31.2 39.72.1 68.33.1 91.71.5 8h 0 0 15.71.5 17.01.7 24.01.7 51.01.7 88.70.6 24 h 0 0 0 12.72.5 17.70.6 34.02.6 88.32.1 48 h 0 0 0 0 11.31.2 20.71.2 86.01.0 72 h 0 0 0 0 6.31.5 15.30.6 83.71.5

32.0 16.0 8.0 4.0 2.0 1.0 Data were expressed as mean SD Control

32.32.5 50.72.3 61.01.7 67.01.7 84.31.2 91.01.0 93.31.2

the extracts tested, rhizomes of A. sivasicum showed the strongest amebicidal effect on the trophozoites (see also Fig. 3). Effect of A. sivasicum rhizome polar extract on the proliferation of E. histolytica trophozoites is presented in Table 1. As can be seen from the table, the extract showed powerful effect on trophozoites. Even in the presence of low extract levels (2.0 mg/ml), no viable trophozoites were observed within the 72nd hour. At 4.0 mg/ml extract concentration, all of the trophozoites available in media have completely been killed by the rhizome extract from the time of the 24th hour. In the presence of the rhizome extracts at 8.0, 16.0, and 32.0 mg/ml concentrations, no viable trophozoites were observed from the time of the 8th hour. At 32.0 mg/ml extract concentration, 73.7% of the trophozoites were successfully killed by the extract within the first experimental hour. Data presented in Table 1 were also analyzed statistically. Except the cells including number 0, data presented in each column and row were found different from each other from the statistical point of view (P <0.05).
Fig. 3 Effect of A. sivasicum rhizomes and aerial parts on the proliferation of E. histolytica trophozoites at 2.0 mg/ml concentration (figure shows the number of viable trophozoites)

Effect of the aerial part extract of A. sivasicum on the proliferation of E. histolytica trophozoites is also presented in Table 2. When compared with the data given in Table 1, the amebicidal effect of this extract was found moderate than that of the rhizome extract. In the presence of 4.0 mg/ml extract concentration, no viable trophozoites were observed within the 48th hour. At 8.0 mg/ml extract concentration, all of the trophozoites available in media have completely been killed by the aerial part extract from the time of the 24th hour. In the presence of the extracts at 16.0 and 32.0 mg/ml concentrations, no viable trophozoites were observed from the time of the 8th hour. At 32.0 mg/ml extract concentration, 67.7% of the trophozoites were successfully killed by the extract within the first experimental hour. Data presented in Table 2 were also analyzed statistically. Except the cells including number 0, data presented in each column and row were again found different from each other from the statistical point of view (P <0.05). For making a better comparison between the results obtained from rhizome and aerial part samples, data obtained from the 2.0and 4.0-mg concentration values (minimum effective

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63 castellani and its cytotoxic potential on corneal cells. J Ocul Pharmacol Ther 24:814 Arrieta J, Reyes B, Calzada F, Cedillo-Rivera R (2001) Amoebicidal and giardicidal compounds from the leaves of Zanthoxylum liebmannianun. Fitoterapia 72:295297 Behnia M, Haghighi A, Komeylizadeh H, Tabaei SJS, Abadi A (2008) Inhibitory effects of Iranian Thymus vulgaris extracts on in vitro growth of Entamoeba histolytica. Korean J Parasitol 46:153156 Block E (1985) The chemistry of garlic and onions. Sci Am 252:114 119 Cao G, Soc E, Prior RL (1996) Antioxidant capacity of tea and common vegetables. J Agric Food Chem 44:34263431 Chu DM, Miles H, Toney D, Ngyuen C, Marciano-Cabral F (1998) Amebicidal activity of plant extracts from Southeast Asia on Acanthamoeba spp. Parasitol Res 84:746752 Davis PH (1984) Flora of Turkey and the East Aegean Islands, vol 8. Edinburgh University Press, Edinburgh Davis PH (1998) Flora of Turkey and the East Aegean Islands, vol 10 (supplement-I). Edinburgh University Press, Edinburgh De Waterbeemd H, Gifford E (2003) Admet in silico modelling: towards prediction paradise. Nat Rev Drug Discov 2:192204 Degerli S, Berk S, Malatyali E, Tepe B (2012) Screening of the in vitro amoebicidal activities of Pastinaca armenea (Fisch. & C.A. Mey.) and Inula oculus-christi (L.) on Acanthamoeba castellani cysts and trophozoites. Parasitol Res. doi:10.1007/s00436-0112524-z Dorant E, Van den Brandt PA, Goldbohm RA, Hermus RJ, Sturmans F (1993) Garlic and its significance for the prevention of cancer in humans: a critical view. Br J Cancer 67:424429 el-Nahas FA, el-Ashmawy MI (2004) Reproductive and cytogenetic toxicity of metronidazole in male mice. Basic Clin Pharmacol Toxicol 94:226231 Gazzani G, Papetti A, Daglia M, Berte F, Gregotti C (1998) Protective activity of water soluble components of some common diet vegetables on rat liver microsome and the effect of thermal treatment. J Agric Food Chem 46:41234127 Gomez M, Perez DG, Ayala P, Samuleson J, Orozco E (1996) Physiology and molecular biology of multidrug resistance in Entamoeba histolytica. Arch Med Res 27:421425 Goze I, Dag S, Tepe B, Akin Polat Z (2009) In vitro amoebicidal activity of Salvia staminea and S. caespitosa on Acanthamoeba castellani and their cytotoxic potentials on corneal cells. J Ocul Pharmacol Ther 25:293298 Guner A, Ozhatay N, Ekim T, Baser KHC (2000) Flora of Turkey and the East Aegean Islands, vol 11 (supplement-II). Edinburgh University Press, Edinburgh Hertog MGL, Kromhout D, Aravanis C, Blackburn H, Buzina R, Fidanza F (1995) Flavonoid intake and longterm risk of coronary heart disease and cancer in the seven countries study. Arch Int Med 155:381386 Hou TJ, Xu XJ (2004) Recent development and application of virtual screening in drug discovery: an overview. Curr Pharm Des 10:10111033 Hou T, Wang J, Zhang W, Xu X (2007) ADME evaluation in drug discovery. 6. Can oral bioavailability in humans be effectively predicted by simple molecular property-based rules? J Chem Inf Model 47:460463 John DT, Petri WA Jr (2006) Markell and Voges medical parasitology, 9th edn. Saunders, Philadelphia, p 23 Kayser O, Kiderlen AF, Croft SL (2003) Natural products as antiparasitic drugs. Parasitol Res 90:5562 Knekt P, Jarvinen R, Reunanen A, Maatela J (1996) Flavonoid intake and coronary mortality in Finland: a cohort study. Br Med J 312:478481 Knemann (1999) Botanica. Gordon Cheers Publication, Hong Kong, p 1020 pp

concentration values for the rhizome and aerial part samples) were also presented in Fig. 3. In the development of drugs intended for oral use, good drug absorption and appropriate drug delivery are very important (Hou and Xu 2004). About 30% of oral drugs fail in development due to their poor pharmacokinetics (De Waterbeemd and Gifford 2003). Among the pharmacokinetic properties, a low and highly variable bioavailability is indeed the main reason for stopping further development of the drug (Hou et al. 2007). Moreover, the knowledge of ionization constant is an important challenge for understanding various phenomena like biological uptake, pharmacological activity, molecule polarity, or in vivo studies (Wani et al. 2011). As can be seen from the experimental part of this paper, methanol extracts obtained from the plant sample were further fractionated with chloroform and water. By this way, polar and nonpolar phytochemicals were separated from the main extract. Biological activity potentials of the polar phytochemicals are well known. Moreover, in some experimental processes, it is necessary using the polar components as the candidate agents due to the polarity of media used. As can be seen from the section mentioned above, the media used in this study require the use of polar substances. These kinds of molecules can easily be integrated into the chemical reactions within the cells and can show desired activity potential. As can be seen from the results presented here, A. sivasicum showed strong activity potential on the proliferation of E. histolytica trophozoites. These results suggest that the plant species evaluated here is a potential therapeutic drug for the treatment of Entamoeba infections. However, further studies to understand the molecular mechanisms involved in the amebicidal actions of these plant species, their pharmacokinetic and pharmacodynamic parameters, active phytochemicals, and their toxicity in animal models are necessary. Drug resistance against metronidazole is still not established, but it may develop in the near future so continuous study is required for herbal extracts and metronidazole.
Acknowledgments This study is financially supported by the Research Council of Cumhuriyet University (CUBAP), SivasTURKEY by the project numbered with F-346.

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