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Anti-Toxoplasma Activities of the Hydroalcoholic Extract of Some Brassicaceae Species

Background: Toxoplasma gondii (T. gondii) is a protozoan parasite that infects a wide range of warm-blooded animals and humans. The conventional anti-Toxoplasma treatments cause significant toxicity. Brassicaceae family contains several medicinal plants with anti-inflammatory, chemopreventive, insecticide, antibacterial, antiviral, and antiparasitic effects. In this study, the hydroalcoholic extract of some Brassicaceae species was investigated against T. gondii in vitro. Materials and Methods: Seeds of Alyssum homolocarpum, Lepidium perfoliatum, Lepidium sativum, and aerial parts of Nasturtium officinale and Capsella bursa-pastoris were extracted by maceration method using 80% ethanol. Vero cells were treated with different concentrations (5–600 μg/mL) of the extracts and pyrimethamine (as positive control), and the cellular viability was verified. Next, Vero cells were infected by T. gondii tachyzoites (RH strain), and the viability of the infected cells was measured by a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Results: The 50% inhibitory concentration values were 5.1, 14.67, 32.49, 37.31, 71.35, and 2.63 μg/mL, and the selectivity indices were 8.06, 2.59, 0.74, 0.78, 0.65 (P < 0.05 compared with positive control), and 3.03 for L. sativum, L. perfoliatum, N. officinale, A. homolocarpum, C. bursa-pastoris, and pyrimethamine, respectively. Conclusion: The results of this study demonstrated that the hydroalcoholic extracts of L. sativum and L. perfoliatum have the promising anti-Toxoplasma activity by growth inhibition of T. gondii tachyzoites in infected cells.


Introduction
Toxoplasma gondii (T. gondii), an obligate intracellular protozoan, infects all mammals. [1] Human can be infected with T. gondii by ingestion of undercooked meat containing tissue cysts and also contaminated water or food with infected cat feces. [2,3] Toxoplasmosis is mostly asymptomatic, but some people may experience flu-like symptoms, swollen lymph glands, malaise, and pains which may last from a few days to several weeks. Toxoplasmosis can be life-threatening in immunocompromised patients. [4,5] In people with HIV infection, toxoplasmosis commonly infects the brain, causes encephalitis, and may also cause death. [6] During pregnancy, T. gondii can be transmitted through the placenta and may result in congenital toxoplasmosis and probably fetal death. Affected infants may suffer from mental retardation, neonatal growth retardation, ocular disorders, and blindness. [7] Combination of pyrimethamine and sulfadiazine is widely used to reduce the risk of clinical manifestation in patients; however, various side effects may occur such as allergic reactions, elevated serum creatinine and liver enzymes, and also bone marrow depression. [8][9][10] In recent years, medicinal plants have been considered as effective and safe sources for drug discovery in the treatment of parasitic diseases. [11] Based on ethnobotanical surveys, it was reported that some Brassicaceae (Cruciferae) plants have been used traditionally for the treatment of parasitic diseases in Iran and other countries. [12][13][14] Moreover, the antiparasitic activities of Brassicaceae have been investigated in recent years. Brassica, Lepidium, Raphanus, and Eruca are the predominant plants in this family with anthelmintic, antitrypanosomal, and antileishmanial activities. [12,[14][15][16] Access this article online Glucosinolates (GSLs) and their hydrolysis products, isothiocyanates, are major groups of naturally occurring compounds found in the seeds, roots, stems, and leaves of Brassicaceae plants. Recently, there are several experimental evidence on the beneficial properties of GSLs against different types of parasites. [17][18][19][20] According to ethnobotanical data and the reported antiparasitic effects from some Brassicaceae species, we aimed to evaluate in vitro anti-Toxoplasma activity of Alyssum homolocarpum, Capsella bursa-pastoris, Lepidium perfoliatum, Lepidium sativum, and Nasturtium officinale hydroalcoholic extracts.

Plant material
The aerial parts of N. officinale and C. bursa-pastoris were collected from North of Iran, Mazandaran, and Golestan Provinces, respectively. A. homolocarpum, L. perfoliatum, and L. sativum seeds were purchased from a local market in Qaemshahr city, Mazandaran province.
The hydroalcoholic extract of the selected plants was obtained by mixing the powdered dry samples with 80% ethanol by maceration method. The resulting extracts were concentrated over a rotary evaporator at 35°C. The remaining semisolid materials were then freeze-dried at −50°C for 24 h and stored at −19°C for further use. For each sample, the stock solution was prepared by dissolving 0.08 g of the extract in 1 mL of dimethyl sulfoxide (DMSO) and diluted with Roswell Park Memorial Institute (RPMI) medium. Finally, it was filtered using a 0.22-micron filter. Different concentrations (5-600 μg/mL) of the extract were obtained by dilution of the stock solution. [21] Toxoplasma gondii strain Six-week-old female BALB/c mice, weighing 18-20 g, were used for this study. All mice were housed in cages under standard laboratory conditions including an average temperature of 20°C-25°C, given drinking water and regular diet according to the Ethics Committee of Mazandaran University of Medical Sciences.
The RH strain of T. gondii was provided by the Toxoplasmosis Research Center in Mazandaran University of Medical Sciences, Sari, Iran. T. gondii tachyzoites were harvested after 3-4 days intraperitoneal injection of 1 × 10 5 parasites to peritoneal cavity of mice. Tachyzoites were suspended in sterile phosphate-buffered saline (pH = 7.4) containing 100 U/mL penicillin and 100 μg/mL streptomycin and counted by hemocytometer under a light microscope (Olympus, Japan). [22]

Effects of plant extracts on intracellular Toxoplasma gondii
For this purpose, Vero cells were cultured in 96-well plates (2 × 10 4 cells/well/180 μL) for 24 h in RPMI 1640 medium supplemented with 10% inactivated FBS at 37°C and 5% CO 2 . Next, the cells were infected with T. gondii tachyzoites (parasite: cell ratio = 10:1). After 24 h, the medium was changed and the infected Vero cells were incubated with different concentrations (5-600 μg/mL) of the extracts. Pyrimethamine (5-600 μg/mL) and RPMI 1640 were also used as positive and negative controls, respectively. MTT solution (5 mg/mL) was added to the cultures and incubated for 4 h. Then, 200 μg/well of DMSO was added to all plates. After 15 min, the optical absorbance was measured at 570 nm wavelength. The growth inhibition concentration was calculated and the mean 50% inhibitory concentration (IC 50 ) was estimated from the dose-response curves of different concentrations of the extracts using the Graph Pad Prism 6.0 software. In addition, Selectivity Index (SI) of the samples was calculated using the IC 50 and the host-cell cytotoxicity profiles (SI = CC 50 /IC 50 ).

Determination of total glucosinolate content
The total GSL contents were evaluated as described by Jezek et al. and Makkar et al. [24,25] The method is based on spectrophotometric evaluation of GSLs after alkaline hydrolysis and reduction with potassium ferricyanide at 420 nm. Sinigrin was used for the preparation of standard curve.
About 500 mg of the powdered seeds was added to 7.5 mL solution of near-boiling acetate buffer (pH 4.2, 0.2 M). The mixture was kept in a boiling water bath (15 min). After cooling, the extracts were mixed with 1.5 mL of barium and lead acetate solution (0.5 M). Then, 0.4 g of polyvinylpolypyrrolidone was added, and the mixture was stirred for 15 min. Finally, 1.5 mL of sodium sulfate solution (2 M) was added, and the mixture was centrifuged (10,000 rpm, 5 min).

Alkaline treatment and reaction with ferricyanide
The clear supernatant was mixed with an equal volume of 2 M sodium hydroxide solution. Concentrated hydrochloric acid (HCL) (37%) was added to neutralize the solution and incubated for 30 min in room temperature. The final mixture was centrifuged (10,000 rpm, 3 min), and the supernatant was mixed with an equal volume of 2 mM ferricyanide prepared in phosphate buffer (pH 7, 0.2 M). The absorbance of the solution was measured quickly against the blank sample.

Preparation of calibration curve
2 M sodium hydroxide solution was added to 500 μL of the sinigrin stock solution (5 mg sinigrin in 1 mL distilled water) and incubated for 30 min at room temperature. Concentrated HCl (37%) was added to neutralize the solution. From this, 0-500 μL was taken and phosphate buffer was added to reach the final volume of 500 μL. After that, 500 μL of potassium ferricyanide solution was added in each tube and mixed thoroughly then centrifuged at 10,000 rpm for 3 min. The supernatant absorbance was measured against the phosphate buffer.

Statistical analysis
Statistical analysis was performed on all data using GraphPad Prism 6.0 software. Differences between the test and control groups were analyzed by analysis of variance and the Newman-Keuls multiple comparison test. P <0.05 was considered statistically significant.

Cellular viability
For evaluating the toxicity of different concentration of the extracts and pyrimethamine on Vero cells in vitro, CC 50 were calculated. The results showed diverse degrees of toxicity on Vero cells. Pyrimethamine was the most toxic treatment for the host cells. The extracts showed less toxicity in comparison with pyrimethamine [ Table 1].

The effects of plant extracts on intracellular Toxoplasma gondii
IC 50 and SI of each extract and pyrimethamine were calculated. Two Lepidum species (L. sativum and L. perfoliatum) showed the best anti-Toxoplasma activity with the IC 50 of 5.1 and 14.67 μg/mL and SIs of 8.06 and 2.59, respectively. The IC 50 and SI of pyrimethamine were determined to be 2.63 μg/mL and 3.03, respectively. The data are shown in Table 1.

Total glucosinolate content
GSLs are one of the main compounds in the seeds of Brassicaceae family. In our study, the seed extracts of L. sativum and L. perfoliatum showed the best anti-Toxoplasma activity. The total GSL content in the seeds of these plants was determined from the sinigrin standard curve and expressed as mmol of GSLs/kg dry weight of seeds [ Table 2].

Discussion
Typically, pyrimethamine alone or combined with sulfadiazine or atovaquone is used to treat T. gondii infections. Treatment with the mentioned drugs, especially pyrimethamine, is associated with toxic side effects such as suppression of the bone marrow, cutaneous rash, leukopenia, and thrombocytopenia. [26] Therefore, research to find effective compounds with less toxicity in the treatment of toxoplasmosis is needed. The antiparasitic activities of some Brassicaceae species have been reported in recent years. [15,16] It is noteworthy that some of the certain species are used commonly in Iran as vegetables or for medicinal purposes. [27] In the present study, the anti-Toxoplasma activity of A. homolocarpum, C. bursa-pastoris, L. perfoliatum, L. sativum, and N. officinale has been investigated for the first time.
According to the results, the SIs of the different extracts were obtained in the following order: L. sativum ˃ L. perfoliatum ˃ N. officinale ˃ A. homolocarpum ˃ C. bursa-pastoris. Thus, the hydroalcoholic extracts of L. sativum and L. perfoliatum were the most effective ones against growth of T. gondii-infected cells (P < 0.05).
Several studies have been conducted on parasitic activity of Lepidium species. In a recent ethnoveterinary study about the treatment of different ailments in dairy animals in Pakistan, some Brassicaceae plants, especially L. sativum, were effective for ectoparasites and endoparasites infections, mastitis, diarrhea, bloating, fever, and anorexia in bovine and bubaline. [14] There is experimental evidence   [28] Recently, a series of novel thiohydantoins (1-benzyl-3-aryl-2-thiohydantoin derivatives) originated from GSLs was isolated from Lepidium meyenii. These compounds showed a variety of activities such as anti-Trypanosoma brucei properties. [29,30] By the comparison of IC 50 and SIs values of the extracts in this study with the results of the previous studies on other plant extracts (Allium paradoxum, Aloe vera, Eucalyptus, Feijoa sellowiana, Quercus castaneifolia, and Sambucus nigra), the seed extract of L. sativum was the most effective (SI: 8.06, IC 50: 5.1 μg/mL, CC 50: 41.11 μg/mL) sample against the growth of Toxoplasma-infected cells with the same methods. [21,31,32] Lepidium species are rich in aromatic GSLs which are converted to isothiocyanates such as benzyl isothiocyanates (BITCs) by hydrolysis. [33] Previous studies indicated that BITCs are the main bioactive compounds responsible for the observed anthelmintic and antinematode activities. [34,35] Steverding et al. reported that dietary isothiocyanates (BITC, phenylethyl isothiocyanate, sulforaphane, erucin, and iberin) have significant in vitro trypanocidal activities against T. brucei. All isothiocyanates showed a dose-dependent effect on the growth of trypanosomes. [15] In our study, the considerable anti-Toxoplasma activity of L. sativum and L. perfoliatum may be related to the presence of GSLs such as BITCs in their seeds.

Conclusion
This is the first report on anti-Toxoplasma activity of some species of the Brassicaceae family. Based on our findings, the hydroalcoholic extracts of L. sativum and L. perfoliatum seeds have the promising anti-Toxoplasma activity by direct growth inhibition of mice cells infected with T. gondii tachyzoites. It seems that Lepidium species can be considered as anti-Toxoplasma agents in future researches.

Financial support and sponsorship
This work was supported by a grant (No. 2804) from research council of Mazandaran University of Medical Sciences, Sari, Iran.