Pak. J. Pharm. Sci., Vol.29, No.6, November 2016, pp.1993-1996 1993 Methanolic extract of Peganum harmala exhibit potent activity against Acanthamoeba castellanii cysts and its encystment in vitro Hafiz Muhammad Shohaib 1 , Salik Nawaz 2 and Abdul Matin 1, 3 * 1 Department of Medical Lab Technology, University of Haripur, Hattar Road, Haripur, Khyber Pakhtunkhwa, Pakistan 2 Department of Pharmaceutical Sciences, University of the Punjab, Lahore, Pakistan 3 Department of Medical Laboratory Sciences, College of Applied Medical Sciences, Majmaah University, Almajmaah, Kingdom of Saudia Arabia Abstract: Acanthamoeba castellanii is member of free living amoeba that may cause painful sight-threatening keratitis and life threatening encephalitis which involves central nervous system. Treatments for both infections are problematic because of the amoebic cysts resistance to therapeutic agents. Here we evaluated in vitro strength of methanolic seed extract of Peganum harmala on Acanthamoeba cysts and its encystment mechanism. Our results revealed seed extracts (1 to 30mg/ml) exhibited amoebicidal effects against Acanthamoeba cysts. Furthermore Acanthamoeba encystment was also inhibited in concentration dependent manner with maximum inhibition at 2µg/ml after 48h incubation. In conclusion, we demonstrated for the first time that methanolic extracts exhibit remarkable inhibition of Acanthamoeba cysts and encystment in vitro which could serve a potential new natural agent against Acanthamoeba. Keywords: Acanthamoeba; amoebicidal effects; encystment; plant extracts. INTRODUCTION Genus Acanthamoeba is member of free-living amoebae that is an opportunistic protozoan pathogen and ubiquitous in nature. Acanthamoeba is known to cause serious human diseases like (i) chronic granulomatous infection involving central nervous system always leading to death, (ii) disseminated infections including skin, sinuses, lungs, prostate and uterus and (iii) painful keratitis (an infection of the eye resulting in blindness) mostly related to contact lens wearers (Trabelsi et al. 2012). Clinical symptoms of Acanthamoeba granulomatous encephalitis (AGE) includes fever, headaches, neurological disorders, personality changes and coma. Acanthamoeba keratitis (AK) is distinguished by ophthalmalgia, photophobia, blue-red vision and blood extravasations. In some cases like the lungs, Acanthamoeba may cause various inflammatory foci (AP), which are escorted by the exudation of serous fluid which carry trophozoites and cysts. In case of skin which changes into various ulcerations. All above mentioned infections are usually chronic. Acanthamoeba lives two stage life cycle i) active trophozoite (infectious stage) and ii) dormant cyst (non- pathogenic or inactive) stage. Acanthamoeba infections are always very difficult to treat and mostly ineffective. Although a number of antimicrobials have been used in the past to treat Acanthamoeba infections but no drug has been found to be useful. There have been few successful treatments reports which were achieved due to the use of combination of drugs against Acanthamoeba trophozoites (Gautom et al., 1998). In spite of this Acanthamoeba eradication from the infection site is very difficult because under unfavorable conditions (i.e. drug treatment), the amoebas trophozoite switch over to cyst stage and medical treatment (drugs) is mostly less effective against amoeba cysts than trophozoites due to the stiff double- wall of the cysts which enables it extreamly resistant to anti-amoebic drugs. This is also problematic as cysts have the capability to resist and survive after initial successful chemotherapeutic administration and cause deterioration of the disease. Encystment is the strategic mechanism amoeba adopts to combat the external threat i.e. harsh environmental conditions and/or drug treatment. The capability of cysts to resist chemotherapeutic agents is perhaps a key factor which contributes the increased cases of protozoan infections. There is an essential need to have a better antimicrobial chemotherapy and alternate strategies to build up therapeutic interventions. The search for new compounds originating from natural resources is an important research area. Many new natural product groups have been identified for antiparasitic properties of with their astonishing efficacy and selectivity such as plant-derived terpenes, alkaloids and phenolics (Kayser et al. 2003). We have previously investigated various plant extracts and reported Peganum harmala have shown the promising effects against Acanthamoeba trophozoites (Shohaib et al., 2013). Therefore use of extracts could have promising outcome in the treatment of Acanthamoeba infections. Here, we investigated efficacy of P. harmala against Acanthamoeba cysts and its encystment mechanism to evaluate a potential therapeutic option of the extract against amoebic encystment using an in vitro model. *Corresponding author: e-mail: a.shafiqurhman@mu.edu.sa