International Journal of Medical Laboratory

Introduction
Acanthamoeba are ubiquitous amphizoic organisms are found in variety of environ-ments including water, sewage, air, soil, food items, swimming pool, dust, dialysis units, humidifiers, and healthy individuals [1-3]. Acanthamoeba keratitis (AK) is a painful sight-threatening ocular disease that occurs generally among immunocompetent individuals. AK symptoms include redness, irritation, tearing, photophobia, ocular pain, lid edema, corneal ring, perineural infiltrates, loose corneal epithelium, and blurred vision [2]. About 85-88% of AK cases are connected with contact lens wearers [4]. The treatment of ocular acanthamoebiasis is extremely difficult and protracted. The combination of cationic antiseptics and aromatic diamidines inhibit membrane functions and DNA synthesis [5, 6].
One of medicinal plants called Myrtus communis is a perennial shrub having a maximum height of 5 meters (m) that belongs to the Myrtaceae family. The leaves of this plant are mostly used in medicine [7]. Myrtus communis extract are useful against different pathogenic organisms and there are good results in terms of antibacterial [8], antiviral [9], bioactivity and antioxidant activity of this plant as well as its antiparasitic [10-12] and antifungal [13] properties. Anti-Acanth-amoeba properties of Myrtus communis are not identified; thus, we focused to explain Anti-Acanthamoeba activity of this extract against trophozoites and cysts of Acanth-amoeba.
Materials and Methods
Preparation of the plant
The dried leaves of Myrtus communis were purchased from the Kerman area in the southeast Iran. The plant was identified and approved by pharmacognosist (Faculty of Pharmacognosy, Shahid Beheshti University, Tehran, Iran).
Extraction of the ethanolic extract
The ethanolic extract of Myrtus communis was obtained by incubating 50 grams (g) of powdered dried leaves in 500 milliliters (ml) of 85-87% ethanol for 3 days. In all cases, the extracts were centrifuged (5000 rpm) for 30 minutes (min) and the supernatants were harvested and filtered using Whatman paper No. 1. The extract was then filtered. Afterwards, a rotary vacuum evaporator at 40°C was used in order to remove the solvent.
Extraction of the aqueous extract
Fifty grams of powdered dried leaves was kept in 200 mL of distilled water for half an hour. Then, the solvent from the extract was removed by rotary evaporator. The residues of extract were collected and kept in the freezer (-20°C) for the experiment.
Acanthamoeba strain
The sample used in this study were obtained from a patient with keratitis. AK diagnosis was based on culture, sequencing analysis and Basic Local Alignment Search Tool (BLAST) search. The sequencing results showed the existence of T4 genotype (accession number KU877552). The specimen was grown on non-nutrient agar (NNA) plates [10] coated with Escherichia coli at 26˚C. The Acanthamoeba strain was kept in non-nutrient agar culture for further use.
Trophozoites
Acanthamoeba was cultivated in NNA medium at 26°C. After 72 to 96 h, trophozoites were washed twice with sterile page’s saline and concentrated at 1500 rpm for 5 min [14]. The number of viable trophozoites were calculated by a neobar lam [15]. The final amount was adjusted to 15×10⁴ trophozoites per ml. Initial cultures were used for testing after 14-21 days and centrifuged at 2000 rpm for 5 min. Cysts were counted and the final amount was adjusted to 15×10⁴ cysts per ml.
Evaluation of activity
In this study, 100 microliters (µl) of the calibrated cyst/ trophozoite suspension (15×10⁴/ml) was inoculated in each micro-centrifuge tube and 100μl of each extract concentration (1.25, 2.5, 5, 10 mg/ml) was added to the tubes. Then, the tubes were kept at 26°C for 24, 48, and 72 hr. In this test, the control tubes included only trophozoites or cysts suspension and sterile distilled water. Three tubes were used for the evaluation of each concentration and measurements were repeated 3 times [16].
Amoebicidial activity of the Myrtus communis on trophozoites and cysts
The samples were incubated with different concentrations of the extract for 24, 48, and 72 h. Then, 25 μl from each test and control well was added into 25 μl from 0.4% trypan blue and the number of live and dead cysts was counted with a neobar lam. This study was approved by the ethical committee of Tarbiat Modares University, Tehran, Iran.
Statistical analysis
The statistical analysis of the data was made by one-way ANOVA and repeated measures tests (compare three or more dependent groups) using SPSS version 18.0. The number of trophozoites and cysts was expressed as mean±SD and percent survival. The significance level was considered as p<0.05.
Results
The results of this research are presented as survival percentage of trophozoites and cysts in Tables 1 and 2. The concentrations of 1.25, 2.5, 5 and 10 mg/ml of aqueous and ethanolic extracts of the Myrtus communis were used against trophozoites and cysts at three different times (24, 48 and 72 hr). The mortality of trophozoites and cysts exposed to the different extracts arised with increase in time of exposure and extract concentration (Fig. 1). The ethanolic extract of Myrtus communis is more effective than the aqueous extract. After adding 10 mg/ml ethanolic extract of Myrtus communis to the medium culture, 0% trophozoites and 8.62% cysts were viable after 48 and 72 hr, respectively. In the case using 5 mg/ml ethanolic extract of Myrtus communis, 28.88% trophozoites and 35.80 % of the cysts were detected in 72 hr. By adding 2.5 mg/ml of extract and after 72 hr  percentages of trophozoites and cysts viabilitywere 43.78% and 56.06%, respectively. Upon administration of extract at concentration of 1.25 mg/ml, 56.98% trophozoites and 75.59% cysts were alive. The difference between these results was statistically significant (p<0.05).
Anti-amoebic activity of extract from Myrtus communis at 10 mg/ml on trophozoites and cysts of Acanthamoeba caused removal of all trophozoites, and 31.10% cysts were viable in 72 h. In the case of 5 mg/ml ethanolic extract of Myrtus communis, 42.22% trophozoites and 63.77 % of the cysts were detected in 72 h. At another concentration, 2.5 mg/ml, 57.33% trophozoites and 69.58% cysts survived. Anti-amoebic activity at 1.25 mg/ml of the extract showed survival of 69.96% of trophozoites and 85.34% of cysts. The difference between the results of the effect of extract on parasite was statistically significant (p<0.05). Finally, among the extracts evaluated, ethanolic extract of Myrtus communis revealed the strongest anti-Acanthamoeba activity on the trophozoites and cysts.

Concentrations  (mg/ml)	Effect on		%Viability of Acanthamoeba
		24th hour	48th hour	72th hour
10.0 (mg/ml)	Trophozoites Cysts	19.99 ±5.44 27.68±4.25	00.00 20.38 ±2.71	00.00 8.62±0.65
5.0 (mg/ml)	Trophozoites Cysts	37.77±3.14 40.00±00	33.33±00 37.68±4.25	28.88±3.14  35.80±5.43
2.5 (mg/ml)	Trophozoites Cysts	48.66±8.37 71.10±7.70	46.66±00 66.50±3.52	43.78±8.80 56.06±3.86
1.25 (mg/ml)	Trophozoites Cysts	64.44±3.13 88.32±2.35	60.31±9.01 76.92 ±00	56.98±6.32 75.59±2.59
Control	Trophozoites Cysts	100±00 100±00	100±00 100±00	100±00 100±00

Concentrations  (mg/ml)	Effect on		%Viability of Acanthamoeba
		24th hour	48th hour	72th hour
10.0 (mg/ml)	Trophozoites Cysts	36.01±9.09 52.22±1.56	33.33±00 46.66 ±6.66	00.00 31.10±3.14
5.0 (mg/ml)	Trophozoites Cysts	54.33±7.93 66.50 ±3.52	48.66±2.01 64.09± 3.62	42.22±3.13 63.77±7.38
2.5 (mg/ml)	Trophozoites Cysts	69.58±2.75 80.16±4.59	65.13±8.55 71.10±7.70	57.33±3.35 69.58±0.09
1.25 (mg/ml)	Trophozoites Cysts	74.52 ±1.69 92.85±7.14	71.10±7.70 86.66±00	69.96±1.03 85.34±0.52
Control	Trophozoites Cysts	100±00 100±00	100±00 100±00	100±00 100±00

AK is an infrequent serious complication in contact lens users that can lead to severe visual loss [17] and promotes with corneal abrasions, poor contact lens hygiene, and home-made saline solutions [2]. The disease is efficintly treated by using antibiotics, propamidine, chlorhexidine, and administering eye drops [18, 19]. Despite combination therapy, only half of the patients have been reported
to improve after treatment with the therapeutic regimens when the disease is not early diagnosed [20]. Failure to treat Acanthamoba infections is due to the following: 1) failure to achieve the optimum dose for the treatment of infections, 2) phenotypic switching (transfor-mation of trophozoite to double-walled cyst), 3) unwanted side effects due to non-selective drugs [21]. Myrtus communis with the common name "myrtle" is a medicinal herb the leaves, branches, berries, and fruits of which have been used widely as a traditional medicine for the treatment of various diseases [22]. The leaves of Myrtus communis are useful in cerebral, stomach and liver diseases, pulmonary disorders, deep sinuses, hair fall, inflammation, haemorrhage, and diarrhea [23]. 
In this context, anti-Acanthamoeba effect
of aqueous and ethanolic extracts of
Myrtus communis against T4 genotype of Acanthamoeba was evaluated. The results of this study showed that aqueous and ethanolic extracts of Myrtus communis on trophozoites are more effective than cysts. Rigid double-layered wall of cyst causes a difference in the sensitivity to drug in the trophozoites and cysts [24].
Many plant extracts have been reported as potent inhibitors of parasites. In the case of Acanthamoeba, different medicinal plants and herbal extracts have been studied as sources of amoebicidal agents. Nayeri Chegeni et al. in 2016 [25] demonstrated that in the presence of 10 mg/ml alcoholic extract in medium culture after 72 hr, 30.51% trophozoites and 91.40% cysts of Acanthamoeba were viable. However, in the presence of 10 mg/ml aqueous extract of Artemisia annua, 58.25% trophozoites and 81.53% cysts were alive in the medium culture after 72 hr. Furthermore, anti-amoebic effects of Peganum harmala ethanolic extract were tested against Acanthamoeba in vitro which ultimately with the effect of 10 mg/ml of extract, 0% trophozoites and 21.10% cysts were identified as alive after 72 hr [26]. Dodangeh et al. revealed that in the presence of 10 mg/ml Chloroformic extract of Trigonella Foenum Graecum, all trophozoites and cysts were removed after 48 and 72 hr, respectively [27]. Polat et al. reported that methanolic extract of Thymus sipyleus subsp was effective on Acanthamoeba castellanii. Finally, 32 mg/ml of the methanolic extract eliminated all trophozoites in 3 h. At the same concentration, no viable cysts were recognized in 12 hr [28]. There are many investigations on effectiveness of plant extracts on parasites that show the importance of natural products for treatment of parasitic disease.
Conclusion
Our findings demonstrated that Myrtus communis inhibits the growth rate of trophozoites and cysts of Acanthamoeba with a dose of 10 mg/ml. It is recommended that cell culture be performed and the extract be used for the animal models in order to determine its exact efficacy and side effects on the human eye.
Conflict of Interest
There is no conflict to declare.
Acknowledgments
We would like to thank the staff of the department of parasitology in Tarbiat Modares University for their help. Also special thanks goes to Dr. Bita Bakhshi, the associate professor of medical bacteriology and Dr. Majid Pirestani assistant professor of medical parasitology in Tarbiat Modares University for their sincere cooperation in this project.

 
 
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