Amebicidal effects of fenugreek (Trigonella foenum‐graecum) against Acanthamoeba cysts

Abstract Trigonella foenum‐graecum L. (TF) is known to the public as a chest emollient, mucous expectorant, laxative and is used to prevent maturation of boils and diabetes since ancient times. In this study, we aimed to determine the amebicidal effects against Acanthamoeba cysts. Plant extracts were prepared at concentrations of 1, 2, 4, 8, 16, and 32 mg/ml and were placed in a hemocytometer with cell counts 22 × 106 cell/ml. The fatty acid profiles of TF seeds were determined. Standard Acanthamoeba cysts were added and incubated at 25°C. The viability of the parasite was checked and recorded at hours 3, 24, 48, 72, 96, and 102. The values of lethal concentration doses (LD50 and LD90) were calculated using probit analysis. This study revealed that T. foenum‐graecum prevented proliferation of the parasite at certain times. However, further for in vivo and controlled experimental studies are needed in order to find out how to use this plant as medication.

Acanthamoeba is among protozoa that have a highly developed structure compared to bacteria. Acanthamoeba is an ameba species that are freely living in natural water resources, sea water, and soil (Saygı & Polat, 2003;Sharma, Garg, & Rao, 2000). This organism has also been isolated from various environments such as swimming pool, tap water, and bottled mineral water, and even in contact lens maintenance water (Saygı & Polat, 2003;Sharma et al., 2000).
In patients with acquired immune deficiency syndrome (AIDS), infection can cause diseases such as chronic sinusitis, otitis, cutaneous lesions, sinus lesions, and skin ulcers due to the spread of the infection to various organs Neelam & Niederkorn, 2017;Niyyati, Lorenzo-Morales, Rezaie, & Rahimi, 2010). Acanthamoeba infection is resistant against numerous antimicrobial agents that can be tolerated in the corneal tissue and ocular surface (Hughes, Andrew, & Kilvington, 2003;Marciano-Cabral & Cabral, 2003;Polat et al., 2008;. It is difficult to treat and eradicate of Acanthamoeba in ophthalmic infections Neelam & Niederkorn, 2017). Although there are many options for the treatment of this infection, these are difficult treatment methods with limited effectiveness (Marciano-Cabral & Cabral, 2003;Tepe et al., 2012). Effective antibiotics for the treatment include propamidine isethionate, ketoconazole, miconazole, itraconazole, and others (Ertabaklar, Dayanır, Apaydın, Ertuğ, & Walochnik, 2009). It has been proven that surgical removal of the lesion with the oral and local administration of miconazole was effective (Ertabaklar et al., 2009). In addition, hydrogen peroxide (H 2 O 2 ) is a commonly used contact lens disinfectant, although is toxic for the cornea (Hughes et al., 2003). Therefore, new approaches and more efficient treatment protocols are needed for Acanthamoeba infections. Today, there are studies conducted with plant extracts and their bioactive compounds in parasitic infections, as in many other scientific areas (Derda, Hadas, & Thiem, 2009;Ródio et al., 2008;Tepe et al., 2012).
Accordingly, we investigated amebicidal effect of the extract on cystic form of the parasite which is reported to be more resistant than the trophozoite form.

| Preparation of Trigonella foenum-graecum (TF) extract
Seed of TF was washed several times with deionized water and dried at room temperature. TF was powdered using a kitchen blender. 100 g of the powdered seeds was added into a 500-ml one-necked flask containing 250 ml methanol, and this was incubated at room temperature (RT: 25°C) for 1 day under stirring. After incubation, each solution was filtered through Whatman filter paper (No. 1) to collect the extract. This step was repeated twice using the same procedure. The extract was collected and evaporated under vacuum at 40°C and then stored at −20°C for further use.

| Determination of the fatty acid (FA) composition of TF
According to the Shimadzu application catalogue, the fatty acid profiles of heat-treated seeds of TF was determined. To investigate fatty acid composition, 2 seeds of TF samples were used for. Fat was extracted with stored in Eppendorf tubes at −20°C until analysis.
The fatty acid composition was analyzed by a gas chromatography (Shimadzu GC-2010 Plus, Japan) equipped with a Flame Ionization Detector and a 100 m × 0.25 mm ID HP-88 column. The injector temperature was set as 250°C. The oven temperature was kept at 103°C for 1 min, then programmed from 103 to 170°C at 6.5°C/min gradient, from 170 to 215°C for 12 min at 2.75°C/min, finally, 230°C for 5 min. The carrier gas was helium with a flow rate of 2 mL/min; the split rate was 1/50. Fatty acid was defined by comparison of retention times with the known standards. The results were expressed as g fatty acid/100 g total fatty acids (Table 1).

| Non-nutrient Agar
Escherichia coli was proliferated on EMB medium prepared according to the procedure. Page's ameba saline solution was used in the study.
The prepared solution was placed in 100 ml Erlenmeyer flasks, autoclaved at 121°C for 15 min, and stored at 4°C until use.

| Preparation of media
Agar of 1.5 g was heated and dissolved in 100 ml Page solution, autoclaved at 121°C for 15 min, and distributed to petri dishes. The prepared media were stored at 4°C until use.

| Culture
The prepared media were diluted with 0.5 ml Page solution, and 24hr E.coli strains were spread on the agar. Samples taken from A. castellanii strains were seeded on the media. The seeded parasites were left at 26°C for 72 hr, and the trophozoites were collected from the petri dishes without harm using Page solution and centrifuged at 1500 g for 5 min for cleaning.
To test the viability of trophozoites, 0.4% trypan blue was used and they were counted on hemocytometer slides.

| Preparation of plant extract concentrations
Plant extract was prepared at concentrations of 32, 16, 8, 4, 2, and 1 mg/ml in 0.9% serum physiologic and distributed to sterile Eppendorf tubes at volumes of 200 μl each.

| Experimental stage
Final concentration of Acanthamoeba castellanii was set to 22 × 10 6 trophozoites/ml, added to the 200μl tubes, and incubated at room temperature. The viability of the parasite was checked and recorded at hours 3, 24, 48, 72, 96, and 102. Tubes with no live cells identified were subjected to control seeding again, and proliferation was not observed in any of these.
Parasites not added to plant extract were left in the same environment as control.

| Statistical analysis
The data were tested for normality using the Shapiro-Wilk test and for homogeneity of variance using the Bartlett's test prior to the analyses. One-way ANOVA followed by Tukey's post-test was used to compare the groups. Descriptive statistics of the data set were expressed as means standard error of mean (SEM). The values of lethal doses (LD 50 and LD 90 ) were determined using probit analysis for the certain times. A p value ≤0.05 was considered statistically significant. All statistical analyses were performed using the SPSS v. 25 (IBM Inc., Chicago, IL, USA) statistical software.

| RE SULTS
The effect of TF (fenugreek) methanol extracts on A. castellanii is given in Table 2. Dead and live ameba are shown in Figure 1.
In our study, there was a decrease in the viability of A. castellaniias time of exposure to T. foenum-graecum extract increased, but there were no statistically significant differences between times in terms of viability rate for the doses of 4, 8, and 32 mg/ml (p > 0.05).
Whereas in the case of T. foenum-graecum extract doses lower or higher than the mentioned doses, there was a statistically significant difference in the rate of A. castellanii viability according to times. As seen in Table 1, there were significant decreases at the 96th hour in the control group, 72th hour at 1 mg/ml dose, and 102th hour at 2 mg/ml (p < 0.05). The rate of viability decreased at a shorter time with increasing the dose of T. foenum-graecum extract to 16 mg/ml, and a significant decrease was observed at the 48th hour (p < 0.05).
While there was no significant difference between 3rd and 24th hours at 16 mg/ml dose of T. foenum-graecum extract (p > 0.05), a significant decrease occurred in the rate of viability at the 48th, 72th, and 96th compared to the 3rd and 24th hours(p < 0.05). In addition, a decrease by about 1/3 was seen in the viability rate after the 48th hours (p < 0.05). However, viability of A. castellanii did not change at the next hours (p > 0.05).

F I G U R E 1 Dead Acanthamoeba castellanii cysts, 40X
was ≤50% in ≥2 mg/ml concentration groups. In the 102th hour, the parasite viability was ≤50% for all plant extract groups including 1 mg/ mL concentration.
In this study also investigated that the LD50 and LD90 value of T. foenum-graecum extract on A. castellanii cysts and trophozoites.
(  sistance of double-walled cysts is due to cellulose molecules found in the inner layer of the cysts. Additionally, the majority of the medications above are very toxic for human keratocytes. The treatment duration for these medications is also very long (may last up to 6 months) (Niyyati et al., 2010;Reinhard & Sundmacher, 2000). Generally, deficiencies of the reported and indicated effective chemotherapeutic agents have lead researchers in the field to give high priority to new compounds for Acanthamoeba infections. In this way, there is a trend of reporting naturally sourced compounds (mainly isolated from plants and herbs) rather than chemical medications (Shinwari, 2010).

| D ISCUSS I ON
Plants produce antimicrobial materials to protect themselves from the pathogenic effects of microorganisms. The main chem- Note. *mg/ml. flavonoids, saponins, fixed oils, and some alkaloids (trigonelline, choline). At the same time, the fenugreek seeds are rich in iron, calcium, phosphorus, and vitamins. Due to the large amounts of galactomannan found in the endosperm of the seed, the plant is thought to increase lactation. Additionally, due to its active components, it has many types of pharmacological activities. Immunomodulatory, anticancer, antidiabetic, gastroprotective, anti-inflammatory, and antipyretic properties of the plant have been identified. Many studies have shown that fenugreek contains defensin (Baldemir & İlgün, 2015;Toppo, Akhand, & Pathak, 2009). Defensin and defensin-like proteins are antifungal proteins and are found in abundant amounts in the seed to protect against soil fungi (Karri & Bharadwaja, 2013;Oddepally & Guruprasad, 2015;Olli & Kirti, 2006).
Some studies have shown that fenugreek seeds are anticancerogenic. They are reported to kill cancer cells from human colon, osteosarcoma, leukemia, lung, and liver cancers. This effect prevents cell growth and is stated to begin apoptosis in a dosedependent manner (Alsemari et al. 2014).
In the present study, we found a significant difference between times of exposure to T. foenum-graecum extract and doses of the  (Dodangeh, Niyyati, & Kamalinejad, 2001).

| CON CLUS ION
This study determined that T. foenum-graecum prevented the proliferation of the parasite at certain times. In addition, we thought that the dose can be increased when a rapid effect of T. foenum-graecum extract on the parasite is desired (LD90 = 36.92 mg/ml), and the dose can be decreased if a long-term effect is expected (LD90 = 16.42 mg/ml) (Table 3). However, further in vivo and controlled experimental studies are needed in order to find out how to use this plant as medication.

ACK N OWLED G EM ENTS
We wish to thank Prof. Dr. Serpil Degerli from Cumhuriyet University for A. castellanii and expert biologist BüşraKir for their help during the experimental stage.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

E TH I C A L S TATEM ENT
Human testing and animal testing were not necessary in this study.