Mohammad Ali Ebrahimzadeh ¹, Mohammad Mohammad Taheri ¹, Ehsan Ahmadpour ², Mahbobeh Montazeri ^3,4, Shahabeddin Sarvi ^3,5, Mohammad Akbari ³, Ahmad Daryani ^{3,5 *}

The currently available agents for use against toxoplasmosis have serious limitations. Thus, the aim of the present study was to investigate the anti-Toxoplasma gondii (T. gondii) activities of methanol extracts of Feijoa sellowiana (F. sellowiana) (leaves and fruits), Quercus castaneifolia (Q. castaneifolia) (fruits), and Allium paradoxum (A. paradoxum) (leaves) in vitro and in vivo.

Vero cells were treated with different concentrations (from 0 to 400 μg/mL) of the above extracts or with pyrimethamine at a dose of 50 mg/mL (positive control). Then, the viabilities of the T. gondii-infected cells were measured by using colorimetric MTT (3-(4, 5-dimethylthiazol-2-yl) 2, 5-diphenyltetrazolium bromide) assays. In addition, the survival rates of mice acutely infected with 2 × 10⁴ RH strain tachyzoites of T. gondii were examined in vivo after intraperitoneal injection of the extracts at doses of 100 and 200 mg/kg day for 5 days.

In the in vitro anti- T. gondii assay, the IC50 values were 12.77, 180.2, 74.73, 213.2 and 163.8 μg/mL, and the selectivity indices were 6.05, 1.31, 0.35, 0.69 and 1.30 for the F. sellowiana (leaves and fruits), Q. castaneifolia, and A. paradoxum extracts and pyrimethamine, respectively. Moreover, the mice treated with F. sellowiana (leaves and fruits) achieved better results in terms of survival than the others (P < 0.05).

The results of the current study indicate that methanol extract of F. sellowiana has significant anti-Toxoplasma activity. Further study should be conducted to investigate the potential bioactivity of this extract through bioactivity-guided fractionation.

Allium paradoxum, Feijoa sellowiana, in vitro, in vivo, Quercus castaneifolia, Toxoplasma gondiiI

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Toxoplasma gondii (T. gondii) is a ubiquitous intracellular protozoan parasite that causes toxoplasmosis in humans and other warm-blooded animals [1]. Globally, over 1 billion people are estimated to be infected with Toxoplasma [2]. Acute toxoplasmosis in immunocompetent people is usually subclinical and asymptomatic, but may lead to chronic infection. However, toxoplasmosis can lead to serious health problems and mortality among immunocompromised individuals, such as human immunodeficiency virus (HIV) positive people, cancer patients, and organ transplant recipients, and congenitally infected neonates [3-6].

The recommended drugs for treatment of toxoplasmosis are pyrimethamine and sulfadiazine [7]. In addition, azithromycin, clarithromycin, spiramycin, dapsone, atovaquone, and cotrimoxazole have been used to treat clinical toxoplasmosis [8-10]. Unfortunately, these atovaquone drugs are poorly tolerated and have no effect on the bradyzoite form [7, 11]. Moreover, the recommended drugs have side effects, such as neutropenia, severe drops in the platelet count, thrombocytopenia, leukopenia, abnormalities, and hypersensitivity reactions [7, 12]. Furthermore, resistance to anti-T. gondii drugs has been reported among acquired immunodeficiency syndrome patients [13]. Accordingly, new drugs with low toxicity and teratogenicity, effective penetration through and concentration in the blood-brain barrier and placenta, parasiticidal effect against the different stage of T. gondii, particularly the cystic form, are urgently needed [14].

In recent years, studies on the therapeutic potential of natural or herbal products have been increasing. Medicinal plants are generally considered to be safe and to have low toxicity compared with synthetic drugs [15]. According to our literature review, the extracts of Artemisia annua L., Glycyrrhiza glabra L., Eurycoma longifolia, Vernonia colorata, Ginkgo biloba, Allium cepa, Zingiber officinale, Myristica fragrans, Astragalus membranaceus Scutellaria baicalensis, Myrrh, Nigella sativa, Sambucus nigra, etc. have been tested for the treatment of toxoplasmosis [16].

Feijoa sellowiana (F. sellowiana) (Myrtaceae) is a small ever green shrub native to the southern areas of South America, where it is extensively distributed. It is widely cultivated in many countries, including Iran [17, 18]. Quercus castaneifolia (Q. castaneifolia) is a plant native to the forests of north, northwest, and west Iran [19]. Allium paradoxum (A. paradoxum), Alezi, is a perennial herbaceous species cultivated in the northern area of Iran, especially in Mazandaran [20]. To the best of our knowledge, we have evaluated in vitro and in vivo for the first time the effects of methanolic extracts from F. sellowiana (leaves and fruits), Q. castaneifolia (fruits), and A. paradoxum (leaves) on T. gondii infection.

This study was performed according to institutional animal ethics guidelines of the Animal Research Center, Mazandaran University of Medical Sciences (ARCMUMS). The animal protocols used in this research were approved by the Mazandaran University of Medical Sciences Ethics Committee (MUMSEC) (Permit number 492).

RPMI-1640 medium, fetal bovine serum (FBS), penicillin- streptomycin solution, and trypsin-EDTA (Sigma, USA) were used for the cell cultures. Dimethyl sulfoxide (DMSO, 99% purity) and MTT (3-(4, 5-dimethylthiazol- 2-yl) 2, 5-diphenyltetrazolium bromide) were purchased from Merck Chemical Company, Germany. Six-week-old, female, inbred Balb/c mice weighing 18 - 20 g were used in this study. The mice were housed in cages (n = 5) under standard laboratory conditions with an average temperature of 20 - 25˚C and were given drinking water and a regular mouse diet [21].

Three types of plants were used in this study: F. sellowiana leaves and fruits, Q. castaneifolia fruits, and A. paradoxum aerial parts were collected from different areas of Mazandaran, Iran, in autumn 2015 and were confirmed by Dr. B. Eslami, Assistant Professor of Plant Systematic and Ecology (Department of Biology, Islamic Azad University of Qhaemshahr, Iran). Voucher specimens were deposited in the Sari School of Pharmacy herbarium (Nos. 194, 195, 991 and A4411). Parts were dried completely in the shade and were triturated in a mechanical mill. The powders were kept in tightened protected containers before use.

To obtain the methanolic extract, we added 150 g of dry powder to 350 mL of pure methanol and used a magnetic stirrer to mix the solution gradually for 1 hour. After the extraction procedure had been completed, the solution was left at room temperature overnight. It was then stirred again and filtered through Whatman No. 1 filter paper, after which the solvent was removed at 40˚C by using a rotary evaporator. The remaining semisolid material was freeze-dried at -50˚C for 24 h. The obtained filtrate (14.5 g) was placed into a sterile glass container and was stored at 4˚C for future use [22]. In order to prepare stock solution (the most concentrated solution), we dissolved 0.02 grams of the extract powders in a minimum amount of RPMI with 10% FBS. A filtration was done using a 0.22-micron filter. Then, the extract was diluted with complete culture medium to a 5-mL volume. Other concentrations (200, 100, 50, 20, 10, and 5 μg/mL) were obtained by dilution of the stock. The solvent was complete culture medium. All processes were performed under sterile conditions. In order to increase the preciseness during this process, we obtained the concentrations in greater volumes than usual.

The RH strain of T. gondii was provided by the Toxoplasmosis Research Center (TRC) in Mazandaran University of Medical Sciences, Sari, Iran. T. gondii tachyzoites were harvested from the peritoneal cavity of Balb/c mice 3 - 4 day after intraperitoneal injection with 1 × 10⁵ of the parasite. The tachyzoites were suspended in sterile phosphate- buffered saline (PBS; pH = 7.4) containing 100 IU/ mL of penicillin and 100 μg/mL of streptomycin [23]. The number of tachyzoites was determined by counting them in a hemacytometer under light microscopy. Vero cells (ATCC No. CCL-81) were used for in vitro assays. Cells were cultivated in RPMI-1640 medium with the addition of 10% inactivated FBS, 100 units/mL of penicillin, and 100 μg/mL of streptomycin. Cultures were kept in disposable cell culture flasks and incubated at 37˚C with 5% CO₂.

The cytotoxicity analysis was done on Vero cells. Cells were seeded in 96-well plates at a density of 1 × 10⁵ cells/ mL suspended in RPMI supplemented with 10% FBS. After 24 h, the cells were exposed to methanol extracts of F. sellowiana leaves, F. sellowiana fruits, Q. castaneifolia fruits, A. paradoxum leaves, and pyrimethamine (positive control) at final concentrations of 5, 10, 20, 50, 100, 200 and 400 μg/mL; the culture medium was used as a negative control. The next day, the viabilities of the Vero cells were evaluated by using a colorimetric MTT assay [24, 25]. Then the 50% cytotoxic concentrations (CC_50s) were calculated by using the Graph Pad Prism 6.0 software (Graph Pad Software, Inc., San Diego, USA).

Vero cells at concentrations of 1 × 10⁵ Vero cells/mL, suspended in RPMI supplemented with 10% FBS, were seeded in 96-well plates. After 8 h of seeding, the cells were infected with the RH strain of T. gondii (1 × 10⁶ tachyzoites/mL) and placed in a 37°C incubator maintained at 5% CO₂ for 24 h. After that, the cells were exposed to the extracts and to pyrimethamine at final concentrations from 0 to 400 μg/ mL; the culture medium was used as a negative control. The next day, the viabilities of the T. gondii-infected Vero cells were evaluated by using MTT assays [24, 25]. Then, the 50% inhibitory concentrations (IC_50s) were calculated by using the Graph Pad Prism 6.0 software. In addition, selectivity indices (SIs) of the drugs were calculated using the IC₅₀ and the host-cell cytotoxicity profiles (SI = CC₅₀/IC₅₀).

Initially, for control of the drugs’ side effects, a preliminary experiment was done on Balb/c mice receiving the same doses of drugs, and the maximum dose at which no mortality or clinically significant toxicity was observed was measured. Then, 50 female Balb/c mice were intraperito-neally inoculated with 2 × 10⁴ tachyzoites of T. gondii RH strain and distributed into 10 groups with 5 animals in each group. On the same day, the mice were intraperitoneally injected with the following extracts and the controls, after which injections were done at regular 24-h intervals for 5 days: F. sellowiana leaves and fruits, Q. castaneifolia fruits, and A. paradoxum leaves extracts at doses of 100 and 200 mg/kg/day, pyrimethamine at a dose of 50 mg/ kg/day (positive control), and PBS (negative control). The survival times were recorded daily until all mice had died.

All tests in this study were performed in triplicate. Statistical analyses were performed using SPSS-14 and Graph Pad Prism 6.0 software. Also, the Kaplan-Meier curve was used to show the survival times, and differences were compared using the log-rank test. Differences were considered statistically significant when P < 0.05.

Treatments of Vero cells with different concentrations of the extracts and pyrimethamine were not toxic compared to the negative control (Fig. 1). However, treatment with F. sellowiana fruit extract and pyrimethamine at a concentration of 400 μg/mL reduced the viability of Vero cells compared to the control while treatments at concentrations from 5 to 200 μg/mL did not affect cell viability. Neither did treatments with F. sellowiana leaf and Q. castaneifolia extracts at doses from 5 to 400 μg/mL. Moreover, cells treated with A. paradoxum extract at concentrations of 200 and 400 μg/mL showed reduced cell viabilities compared to the control while those treated at concentrations from 5 to 100 μg/mL showed no effect on cell viability.

In this study, the growth inhibitions of T. gondii in infected cells were measured at different concentrations of the F. sellowiana, Q. castaneifolia, and A. paradoxum extracts and pyrimethamine. F. sellowiana leaves showed anti-T. gondii activity with an IC₅₀ of 12.77 and SI of 6.05. In addition, pyrimethamine’s activity was characterized by an IC₅₀ of 163.8 and SI of 1.33 (Table 1).

Clinically, the numbers of mice in the untreated infected groups (negative control) started to decrease on the seventh day of the study, and all mice had died by the end of the eighth day. Mice in the other treatment groups started to die on the eighth day, and all mice had died by the end of the eleventh day. The treatment with F. sellowiana leaves achieved better survival results in the mice than treatments with F. sellowiana fruits, Q. castaneifolia, and A. paradoxum did (Fig. 2). Mice in the groups treated with F. sellowiana fruits at a dose of 100 mg/kg/day and with F. sellowiana leaves and A. paradoxum at doses of 100 and 200 mg/kg showed statistically significant higher survival rates than the mice in the untreated infected control group (P < 0.05). Statistically significant differences were observed among the group treated with F. sellowiana fruit at a dose of 100 mg/kg/day), the groups treated with F. sellowiana leaves, Q. castaneifolia, and A. paradoxum at a dose of 200 mg/kg/day), and the positive control (P < 0.05).

The standard anti-T. gondii chemotherapy is still limited as it is not well-tolerated by immunocompromised patients and cannot completely eradicate tissue cysts produced by the parasite [14]. Thus, new, less toxic compounds for use against Toxoplasma are critically needed [12, 26]. Natural compounds and traditional herbal medicine have high availability and lower side effects compared with current chemical drugs [16]. This study was, therefore, performed to evaluate in vitro and in vivo the activities of methanol extracts of F. sellowiana, Q. castaneifolia, and A. paradoxum against T. gondii. To the best of our knowledge, such a study has not been previously reported in the literature.

In our in vitro results, the anti-T. gondii activity of the agents was calculated as the selectivity index. The selectivity index was indirectly applied to the cell viability of Vero cells to represent the parasite infection ratio. According to the results, the SIs of the different compounds were obtained in the following order: F. sellowiana leaves > F. sellowiana fruits > pyrimethamine > A. paradoxum leaves > Q. castaneifolia fruits. Thus, F. sellowiana, especially F. sellowiana leaves, showed an anti-T. gondii activity against T. gondii-infected Vero cells higher than that of the standard treatment.

F. sellowiana has multi-pharmacological effects, including antimicrobial, antifungal, antioxidation, antidepressant and anti-cancer activities, which has been confirmed by several researchers [17, 18, 27, 28]. The leaves and the fruits of F. sellowiana contain many basic components, such as flavonoids, tannins, terpenes, and steroidal saponins [29]. Similarly, Zhang et al showed that the activities and SIs of oxymatrine and matrine, the main alkaloids in sophora leguminous plants, were higher than that of spiramycin against the RH strain of T. gondii-infected HeLa cells [30].

In our study, the effects of F. sellowiana, Q. castaneifolia, and A. paradoxum were analyzed in acute infections with T. gondii RH strain in Balb/c mice in vivo. Given that a concentration of 400 μg/mL was found to be toxic in in vitro studies on some extracts, we selected concentrations of 100 and 200 mg/kg/day for our in vivo study. Treatment of experimental mice with F. sellowiana and pyrimethamine for 5 days after infection with 2 × 10⁴ tachyzoites of the T. gondii RH strain statistically increased their survival rates when compared to mice in the untreated infected control. Although, the mice treated with methanol extract of F. sellowiana leaves (200 mg/kg/day) exhibited better survival than the mice treated with the other methanol extracts, the differences in survival between those mice and the mice in either the positive or the negative control groups were not statistically significant, indicating that that the activity of F. sellowiana is probably due to its high antioxidation properties and its high phenolic and flavonoid contents [17]. Keles et al demonstrated that F. sellowiana extracts displayed remarkable antioxidant activity and decreased lipid peroxidation in rats [29].

Moreover, Q. castaneifolia increased the survival rate of the mice compared to that of the mice in the untreated infected control, and the differences compared to F. sellowiana leaves (200 mg/kg/day) and pyrimethamine were statistically significant. Q. castaneifolia fruit extract contains many basic components and has antimicrobial effects on the E. coli, salmonella typhimurium, Shigella dysenteriae and Yersinia enterocolitica [31]. Also, a significant difference existed between A. paradoxum and the untreated group (negative control) and the group treated with F. sellowiana fruits (200 mg/kg/day). Interestingly, Raeisi and Rahimi Esboei showed that A. paradoxum was not significantly effective during hydatid cyst treatment at concentrations of 1, 10, 50 and 100 mg/mL [32].

A. paradoxum has been reported to be an Iranian native plant that exhibits antimicrobial, antifungal, and antiviral effects. Also, the aerial extracts of A. paradoxum have been reported to exhibit antioxidant activity [31]. B-Pinene, limonene, Z-nerolidol, spathulenol, alpha-bisabolol, phytol, n-docosane and n-tricosane are the most important reported chemical components of A. paradoxum [31]. In an in vivo study, Hezarjaribi et al found that an ethanol extract of A. paradoxum at a concentration of 100 mg/mL was more effective for treating Giardia cysts than the chloroform extract [33]. In addition, Elmi showed that the ethanol extract of A. paradoxum was significantly more effective than the chloroform extract in the treatment of mice infected with Giardia parasites [34]. Our present study showed that the methanol extract of A. paradoxum exhibited no significant anti-Toxoplasma activity in vitro and in vivo. Based on these results, we hypothesize that the ethanol, liquid, and chloroform extracts of A. paradoxum may be more active against toxoplasma infection than the methanol extract.

In conclusion, this is the first report on the anti-Toxoplasma activities of methanol extracts of F. sellowiana, Q. castaneifolia, and A. paradoxum. Given that the Q. castaneifolia extract at the concentration used in this study had no toxicity in the in vitro study, higher concentrations should be used in future studies to evaluate its therapeutic potential in vitro and in vitro. The results of the current study indicate that F. sellowiana promotes significant activity against T. gondii. Further study should be designed to investigate the potential bioactivity of this extract through bioactivity-guided fractionation and to identify the additional mechanisms involved in its effects.

We thank Dr. Mohammad Taghi Rahimi, School of Medicine, Shahroud University of Medical Sciences, Shahroud, Iran, for support with the in vivo study. We acknowledge the financial support (grant no. 492) from the Mazandaran University of Medical Science, Iran.

The authors declare that they have no conflicts of interest.

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