Anti‐Toxoplasma activity and chemical compositions of aquatic extract of Mentha pulegium L. and Rubus idaeus L.: An in vitro study

Abstract This study aimed to determine the chemical compositions of crude aquatic extracts of M. pulegium L. and R. idaeus L., and their anti‐Toxoplasma activity. Crude aquatic extraction of aerial parts of R. idaeus L. and M. pulegium L. was performed. GC‐MS and HTPLC analyses were carried out. MTT assay was performed on Vero cells treated by different concentrations (Log −10 from 10−1 to 10−6) of the extracts. The anti‐Toxoplasma activity of the concentrations was investigated using vital staining. Menthol (99.23%) and limonene (0.227%) were the major compounds of the aquatic extract of M. pulegium L. Phytochemical compositions of R. idaeus L. were terpenoids, esterols, and flavonoids. The cell toxicity of M. pulegium L. was lower than R. idaeus L. (CC50 > 10−2 versus. ≥ 10−4). Aquatic extract of M. pulegium L. showed higher anti‐Toxoplasma activity (LC50 ≥ 10−6) than R. idaeus L. (LC50 ≥ 10−5). Statistically significant cell toxicity and anti‐Toxoplasma activity (p < .05) were seen regarding the different concentrations of R. idaeus L. and M. pulegium L. Both R. idaeus L. and M. pulegium L. revealed anti‐Toxoplasma activities. Cell toxicity of R. idaeus L. was significantly higher than M. pulegium L. M. pulegium L. extract could be more applicable due to its lower cell toxicity.


| INTRODUC TI ON
Toxoplasmosis caused by an intracellular parasite, Toxoplasma gondii, is frequently reported from almost all countries (Montoya & Liesenfeld, 2004;Robert-Gangneux & Darde, 2012). The infection may occur via vertical transmission from infected mothers, white blood cell (WBC) transfusion, transplantation, consumption of contaminated food and water, and close contact to infected cats (Hide, 2016;Hill & Dubey, 2002, 2016. Tachyzoite, bradyzoite, and oocyst are the main three forms of Toxoplasma's life cycle. Tachyzoites are responsible for the acute phase of toxoplasmosis while bradyzoites are seen in tissue cysts and chronic phase of infection (Montoya & Liesenfeld, 2004).
Currently, a panel of antibiotics is being recommended and widely practiced for both chronic and acute toxoplasmosis (Alday & Doggett, 2017). Accordingly, a combination of sulfadiazine and pyrimethamine is known as a standard antibiotic therapy for toxoplasmosis (Alday & Doggett, 2017;Montazeri et al., 2017). Moreover, clindamycin, pentamidine, atovaquone, and azithromycin are another drugs of choice which are widely prescribed in the clinical practices (Alday & Doggett, 2017). However, low bioavailability, need for a high dosage, and wide side effects including bone marrow suppression, diarrhea, and abdominal pain limit prescription of these drugs (Alday & Doggett, 2017;Darade, Pathak, Sharma, & Patravale, 2018).
During the recent decades, herbal medicine has been explained and practiced as an alternative therapy with low toxicity for a broad range of infective and noninfective diseases (Choi, Gang, & Yun, 2008;Kheirandish et al., 2016;Seo et al., 2019;Yadav & Temjenmongla, 2012;Yeom et al., 2015). Mentha belongs to Labaiatae family (Bhat, Maheshwari, Kumar, & Kumar, 2002) and is found all over the world. Mentha pulegium L. is a species of this genus that its antimicrobial effects have been evaluated on bacteria and protozoa (Bouyahya et al., 2017;Mahboubi & Haghi, 2008). Rubus idaeus L. belongs to Rubus genus and Rosaceae family, mainly grows in the north regions of Iran (Moreno-Medina, Casierra-Posada, & Cutler, 2018;Nalbandi, Seiiedlou, Hajilou, & Adlipour, 2011). Although many studies investigated the anticancer, anti-inflammation, and antimicrobial effects of the genus Rubus (Krauze- Seo et al., 2019), there is no available data suggesting anti-parasitic effects of this plant.
In the current study, chemical compositions of crude aquatic extracts of M. pulegium L. and R. idaeus L. and their anti-Toxoplasma were evaluated.

| Extract preparation
R. idaeus L. and M. pulegium L. aerial parts were hand-picked on same day from a local farm in the northern parts of Iran in August 2016.
Plants were air-dried in 25°C, away from sunshine, with continuous air ventilation until a constant weight obtained. Extraction was performed on powdered dry plant using decoction technique. Briefly, 500 g of plants were subjected to 2 L of distilled water and boiled until the volume decreased to its 1/4. Then, the mixtures were filtered and the extracts were concentrated using indirect heat.

| Gas chromatography (GC)/mass spectrometry (MS)
Crude aquatic extracts of R. idaeus L. and M. pulegium L. were subjected to a solvent-solvent partitioning with petroleum ether, chloroform, and ethanol. Main components existing in resulted fractions were identified by GC-MS using a HP-5ms column (30 m × 0.25 mm, film thickness 0.25 μm; Agilent Technology).
Temperature of the column was maintained at 50˚C for 1 min and programmed to 250˚C at a rate of 3˚C per min, and was constant at 250˚C for 20 min. Injector and detector temperatures were 250˚C and 230˚C, respectively. The flow rate of the carrier gas was 1 ml/ min. Helium 99.999% was used as the carrier gas with ionization voltage of 70 eV. Mass range was from 40 to 400 u. Identification of the components was performed by comparison of their relative retention time and mass spectra to the standards (Wiley 7 library data of the GC-MS system).

Solvent extraction
In order to cover a wide range of polarity, 0.1 g of R. idaeus L. total extract was subjected to a serial extraction with four organic solvents, starting from an absolutely nonpolar solvent (hexane) followed by gradually increasing polarity solvents (ethyl acetate, chloroform, methanol). Next, acid and alkaline extraction were performed on methanol components by using HCl 0.1 M and NH 3 10% v/v. The final extraction of salts was accomplished with chloroform.

Chromatographic experiment and phytochemical screening
HPTLC is illustrative for expansion of chromatographic fingerprints to determine major active constituents of medicinal plants. It also presented a more efficient separation of individual secondary metabolites. 10 µl of each sample solutions were applied on the TLC plate using ATS 4 in the form of band (band width: 6 mm, distance between two bands: 9.4 mm). A constant application rate of 150 nl/s was used with the mobile phase of chloroform-methanol (8:2) v/v.
The plates were then placed in the mobile phase, and ascending development was performed to a distance of 7 cm. Subsequent to the development, the plates were air-dried and chromatograms were evaluated with TLC visualizer under 254 nm and under 366nm, and white light.
Qualitative TLC analysis was performed. Accordingly, chloroform-methanol (7:3) was the mobile phase system and silica gel 60 F 254 HPTLC plate was incorporated as stationary phase. Five different reagents including anisaldehyde sulfuric acid, dragendorff, ninhydrin, potassium hydroxide, and sulfuric acid were utilized to describe the secondary metabolic compounds (terpenoids, saponins, alkaloids, amino acids, antraquinones), which were found in extracts.
The plate was documented in daylight and at UV 366 nm mode using the photo-documentation chamber.

| T. Gondii strain
At the first step, the RH strain of T. gondii was kindly supplied by Dr. SJ Seyed Tabaei from intraperitoneal (IP) passages of Toxoplasma in female BALB/c mice (8-to 10-week-old, 20-25 g weight), 3-4 day after IP injection with 1 × 10 7 of the parasite.
All mice were housed in cages under standard laboratory conditions including an average temperature (20-25°C), humidity (60 ± 10%), light (12 hr per day), given drinking water, and regular diet in the animal center of Shahid Beheshti University of Medical Sciences of Tehran, Iran.
The tachyzoites were collected from peritoneal cavity of infected mice. Afterward, the parasites were washed using sterile PBS (pH: 7.4), counted by hemocytometer slide, and 1.5 × 10 6 tachyzoites were inoculated to the Vero cells, kidney fibroblast from the African green monkey, cultivated in dulbecco's modified eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin for mass-cultivation.

| Cell culture
Vero cells were used for in vitro assays. In this regard, the cells were cultivated in DMEM medium, supplemented with 10% heat-inactivated FBS and 1% penicillin and streptomycin. Cultured media were maintained at 37°C in 5% CO 2 . When the cells reach to 80% confluent, sub-culture was performed.

| Cell toxicity assay for Vero cell
To evaluate the cell toxicity of the herbs, Vero cells were seeded in 96-well plates (cell suspensions 2.4 × 10 5 cell/mL in complete culture medium DMEM and incubated at 37°C in 5% CO 2 for 48 hr). After 48 hr, serial dilutions 10 −1 to 10 −6 of each R. idaeus L. and M. pulegium L. extract were added and incubated at 37°C in 5% CO 2 for 2 days.
After 48 hr, the cell viability was measured by adding MTT solution (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) to the cultures. A well of Vero cell without treatment was considered as negative control. After 4 hr, the experiment was stopped by DMSO and the results were read at wavelength 590 using an enzyme-linked immunosorbent assay (ELISA) microplate reader (LX800; Biotec, Winooski, VA, USA). All experiments were performed in duplicate.
The nonviable Vero cells were calculated using the following equation , and the 50% cytotoxic concentrations (CC50s) were calculated using the Graph Pad Prism 6.0 software (Graph Pad Software, Inc., San Diego, USA).
AT is the OD of treated well, AC is the OD of negative control, and AB is the OD of the blank well.

| Effect of the plant extracts on tachyzoites of Toxoplasma
To evaluate the toxicity of the extracts, 1 × 10 6 tachyzoites per well of T. gondii were added to a 96-well plate containing DMEM supplemented with 10% FBS without antibiotics; then, serial dilutions 10 −1 to 10 −6 of the extracts were added to wells. After 2 hr, 10 µl of T. gondii from each well containing different concentrations of the extracts was stained by trypan blue, and the number of alive cells was calculated using hemocytometer slide and optical microscopy. A well containing T. gondii without any herbal extract was considered as negative control. All tests performed in duplicate.

| Statistical analysis
Statistical analyses were performed on all data using Graph Pad Prism (version 6.07) software. Differences between test and control groups were analyzed by one-sample t test. p < .05 was considered as statistically significant.

| GC-MS analysis
In all three fractions of M. pulegium L., menthol and limonene were identified as two components ( Table 1)

| Phytochemistry and HPTLC analyses
HPTLC presented the fingerprint of R. idaeus extract's constituents.
Constituents were individualized due to a serial extraction of total extract with hexane, ethyl acetate, chloroform, methanol, HCl, and NH 3 as specified spots on TLC paper, under white light, and UV light (366 and 254 nm) (Figure 1). Preliminary phytochemical analysis obtained from exposure of R. idaeus fingerprint to secondary metabolite reagents (anisaldehyde sulfuric acid, dragendorff`s, ninhydrin, potassium hydroxide, and sulfuric acid) revealed the presence of terpenoids, esterols, and flavonoids based on color zones obtained ( Figure 2).

| Cell viability of Vero in different concentrations of M. pulegium L. and R. idaeus L. extracts
To analyze the toxicity of extracts on host cells, we investigated cell viability of Vero cell treated with M. pulegium L. and R. idaeus L. at the concentrations ranging from 10 −1 to 10 −6 using the MTT assay.

| R. idaeus L
MTT results showed that R. idaeus L. extract significantly reduced cell viability of Vero cells in concentrations more than 10 −5 .
Accordingly, the results showed that the CC50 of R. idaeus L. was about 10 −4 (CC50 ≥ 10 −4 ). The results showed that in the highest  Figure 3a; Table 3).

| Effects of M. pulegium L. and R. idaeus L.extracts on T. gondii
To analyze the anti-Toxoplasma effects of each extract, different concentrations of M. pulegium L. and R. idaeus L. extracts were examined.

| Ratio analysis
In order to calculate the best concentration with highest anti-Toxoplasma activity and lowest cell toxicity, ratio analysis was performed. The value closer to 1 considered as the recommended value.
Accordingly, 10 −4 with ratio 1.08 was the suggested concentration with highest and lowest anti-Toxoplasma and cell toxicity, respectively, for M. pulegium L. The concentration 10 −5 of R. idaeus L. with ratio 0.86 seems to be the best concentration.

| D ISCUSS I ON
During recent years, interest on the traditional drugs, particularly herbal medicine, is being dramatically increased (Kheirandish et al., 2016). It seems that lower side effects and costs are the main reasons of this interest. Plenty of studies have practiced different herbal extracts on both helminths (Dejani et al., 2014;Yadav & Tangpu, 2008Yadav & Temjenmongla, 2012) and protozoa (Dyab, Yones, Ibraheim, & Hassan, 2016;Kheirandish et al., 2016;Mirzaalizadeh et al., 2018;Ribeiro et al., 2014)  However, the antimicrobial effects of M. pulegium L. and its compounds have been widely practiced. Mahboubi and Haghi (2008) showed that the M. pulegium L. essential oil extract had significant antimicrobial effects, particularly on the Gram-positive bacteria.
In studies conducted by Dejani et al. (2014) and Zaia et al., (2016), the anti-Schistosoma (S. mansoni) and anti-inflammatory effects of crude ethanol extract and menthol from M. piperita L., respectively, were investigated that the findings were promising. In another study by Bouyahya et al. (2017), pulegone and menthone were characterized as the major compounds of M. pulegium which showed F I G U R E 3 Ratio analyses of (a) R. idaeus L. and (b) M. pulegium L. show anti-Toxoplasma and cell toxicity of the extracts. CC50 and LC50 for R. idaeus L. are at the concentrations more than 10 −4 and 10 −5 , respectively, while CC50 for M. pulegium L. is more than 10 −2 , and LC50 is ≥ 10 −6 F I G U R E 2 Phytochemical analysis of R. idaeus L. extract under (a) visible and (b) UV light with secondary metabolite reagents: 1-anisaldehyde sulfuric acid, 2-dragendorff`s, 3-natural products, 4-ninhydrin, 5-potassium hydroxide, and 6-sulfuric acid

| CON CLUS ION
According to our findings, both R. idaeus L. and M. pulegium L. revealed anti-Toxoplasma activity. In addition, cytotoxicity activity of R. idaeus L. was significantly higher than that in M. pulegium L.
Therefore, although anti-Toxoplasma activity of both R. idaeus L. and M. pulegium L. extracts might be promising, our results showed that M. pulegium L. extract could be more applicable due to its lower cell toxicity in higher concentration in comparison to R. idaeus L.