Evaluation of Origanum vulgare Essential Oil and Its Active Ingredients as Potential Drugs for the Treatment of Toxoplasmosis

Toxoplasma gondii is a serious hazard to public health and animal husbandry. Due to the current dilemma of treatment of toxoplasmosis, it is urgent to find new anti-T. gondii drugs to treat toxoplasmosis. In this study, the anti-T. gondii activity of Origanum vulgare essential oil (Ov EO) was firstly studied, and then, carvanol (Ca), the main ingredient of Ov EO was evaluated using the MTT assay on human foreskin fibroblast (HFF) cells in vitro. The cytotoxicity was evaluated using the MTT assay on HFF cells. The CC50 of Ov EO and Ca was 134.9 and 43.93 μg/ml, respectively. Both of them exhibited anti-parasitic activity, and inhibited the growth of T. gondii in a dose-dependent manner. For the inhibition effect, Ca was better than Ov EO at the same concentration, the IC50 of Ov EO and Ca was 16.08 and 7.688 μg/ml, respectively. In addition, treatment with Ca, was found to change the morphology of T. gondii tachyzoites and made their shapes curl up. These results showed that Ca was able to inhibit the proliferation of T. gondii by reducing invasion, which may be due to its detrimental effect on the mobility of tachyzoites. Our results indicated that Ca could be a potential new and effective drug for treating toxoplasmosis.


INTRODUCTION
The opportunistic pathogen Toxoplasma gondii is a serious hazard to public health and animal husbandry (Chemoh et al., 2013). One-third of the people in the world have been infected by T. gondii where tachyzoites, cysts and oocysts are three infectious stages. Human intake of raw meat or water containing T. gondii cysts or oocysts can be infectious. In a few cases, direct contact with T. gondii tachyzoites between the mucous membrane and the damaged skin can also cause an infection. Cats are intermediate hosts of T. gondii and can rule out infectious oocysts. Accidental contact between humans and cat feces is a risk of infection. For most individuals with competent immunity, infection is asymptomatic and the T. gondii eventually lies dormant as a tissue cyst. For some people primary infection can cause ocular disease, and in pregnant women, it can lead to abortion, stillbirth or brain damage in a congenitally infected fetus. Recurrence of chronic infection is a frequent cause of toxoplasmic encephalitis (TE) in an immunosuppressive patient such as advanced HIV infection, neoplastic disease, or in those receiving immunosuppressive therapies (e.g., rituximab).
As T. gondii has a wide range of hosts, apart from humans, it also infects many animals, like cattle, sheep, pig and other domestic animals. This causes the economic loss of animal husbandry and the hidden danger of public food hygiene and safety.
The drug treatment of toxoplasmosis can be traced back to the use of sulfonamides in the 1940s. In the 1950s, sulfadiazine combined with pyrimethamine successfully treated toxoplasmosis in mice. It is still the golden treatment for toxoplasmosis today (Wei et al., 2015). However, the side effects and the emergence of drug resistance have undermined the perfection of the treatment regimen (Schmidt et al., 2006). In the case of pregnant women infected with T. gondii, spiramycin is a good drug for the treatment of toxoplasmosis because of its low toxicity and it cannot penetrate the placental barrier; however it has no effect on the infected fetus (Desmonts and Couvreur, 1974). Other drugs such as Trimethoprimsulfamethoxazole, Clindamycin, and Atovaquone also have their own disadvantages (Dunay et al., 2018). Therefore, it is urgent to find new anti-T. gondii drugs with high efficiency and low toxicity to treat toxoplasmosis.
Natural products are one of the important sources of drug development (Petrovska, 2012). In the field of cancer treatment alone, from the 1940s to now, 48.6% of the 175 small molecules are natural products or obtained directly from there (Newman and Cragg, 2012). Plants as one of the natural products usually grow outdoors, so they have to resist the infection of disease and the pressure of harsh environment in the process of growing (Weng et al., 2012). In this process of defense, the molecules they produce give plants smell, color and even toxicity (Lietava, 1992). Essential oils are a mixture of these molecules, which are a potential drug reservoir.
Origanum vulgare that is native to the Mediterranean coast, North Africa and West Asia is a perennial herb of the genus Oregon of the Lamiaceae family (Elshafie et al., 2017). The O. vulgare essential oil (Ov EO) has been proven to have certain biological activity (Argyri et al., 2021). At a concentration of 60 mg/ml, Ov EO can inhibit the invasion rate of Cryptosporidium parvum into Human colon adenocarcinoma (HCT-8) cells by 60% (Gaur et al., 2018). Ov EO can also inhibit the growth of Aeromonas hydrophila, Brevibacterium linens, Clostridium sporogenes, Leuconostoc cremoris, and Pseudomonas aeruginosa (Dorman and Deans, 2000). The crude Ov EO can decrease the activity of liver cancer cells (HepG2 cell) in a dose-dependent manner, and the IC50 value was 236 mg/ml (Elshafie et al., 2017). In addition, Ov EO also shows excellent anti-inflammatory and antioxidant activities, and also has potential functions in controlling cardiovascular diseases and metabolic syndrome (Leyva-Loṕez et al., 2017).
In this study, the anti-T. gondii activity of Ov EO was firstly studied, and then the inhibited activity of carvanol (Ca), the main ingredient of Ov EO, was selected to evaluate in vitro.

Cell Culture and Parasites
T. gondii tachyzoites of the GFP-RH strain, expressing green fluorescence protein were proliferated in human foreskin fibroblast (HFF) cells, cultured in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% heat-inactivated fetal bovine serum (FBS) at 37°C, in an atmosphere containing 5% CO 2 . To isolate the tachyzoites, heavily infected cells were scraped and the parasites were released by passing the cells through a 27-gauge needle, three to five times. Cell debris was removed by passing the mixture through a 3-µm pore membrane filter (Whatman, ThermoFisher, Waltham, MA, USA). Tachyzoites were quantified using a hemocytometer before proceeding to further experiments.

Essential Oil and Chemical Components
The Ov EO and Ca used in this experiment was provided by Guangxi University, EO was extracted by steam distillation and dissolved in dimethyl sulfoxide (DMSO) in a 1:1 ratio. Ca was dissolved into a suitable mother liquor with DMSO. The species number of O. vulgare used in this study is GXCM 2019023. The solutions were then diluted with DMEM, such that the final concentration of DMSO in the samples used in the experiment was lower than 1.56% v/v.

Cytotoxicity Assay
The cytotoxicity of Ov EO and Ca was evaluated in an HFF cell line with a CellTiter 96 ® AQueous One Solution Cell Proliferation Assay (Promega Corp., Madison, WI, USA), according to the manufacturer's instructions. HFF cells (1 × 10 5 cells/well) were seeded in 96-well plates and cultured at 37°C, in an atmosphere containing 5% CO 2 , for 24 h. The cells were treated with varying concentrations of Ov EO (70,35,17,9,and 4 mg/ml),Ca (70,35,17,9,and 4mg/ml) or sulfamethoxazole (SMZ), and a 1.56% solution of DMSO in DMEM was used as the vehicle control. After incubating for 24 h, the HFF cells viability were measured by the MTT (3-[4,5-methylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) colorimetric method according to Costa et al. (2018). Approximately 20 ml of MTT solution (5 mg/ml) was added to each well and allowed to incubate at 37°C with 5% CO 2 for 3 h and then 200 ml of DMSO was added to dissolve the formazan crystals. Absorbance was measured at 490 nm using an iMark ™ Microplate Absorbance Reader (BioRad, Hercules, CA, USA). and the 50% cytotoxic concentrations (CC 50 ) were calculated using Graph Pad Prism 8.0. The cytotoxicity experiment was performed in triplicate, using three separate plates.

Anti-T. gondii Activity of Ov EO and Ca Evaluated by a Plaque Assay
One hundred freshly released GFP-RH tachyzoites were added to HFF monolayers in 6-well plates, in DMEM with 2% FBS. They were incubated at 37°C, in an atmosphere containing 5% CO 2 , for 4 h. Then, the extracellular parasites were removed with medium, and fresh medium containing various concentrations of Ov EOs, Ca or 1.56% DMSO (vehicle control) were added to each well. Uninfected and untreated wells were used as blank controls. After 7 days, HFF cells were washed three times with PBS, fixed with methanol for 10 min, and stained with 0.1% crystal violet for 30 min. After washing three times with phosphate buffered saline (PBS) and drying naturally (Huang et al., 2021), the plaques formed by tachyzoites were examined by microscopy.
Anti-T. gondii Activity of Ov EO Evaluated by an Intracellular Growth Assay HFF cells were incubated in 24-well plates for 48 h, then the medium was replaced by DMEM with 2% FBS, 100 freshly released GFP-RH tachyzoites were added to each well, and incubated at 37°C in an atmosphere containing 5% CO 2 , for 4 h. The medium containing extracellular parasites was removed and fresh medium containing either Ov EO (70,35,17,9,and 4 mg/ml) or Ca (17,9,4, and 2 mg/ml), 1.56% DMSO (vehicle control), or 10 mg/ml SMZ (positive control) was added to each well. After 32 h, the growth of GFP-RH was observed and photographed under a fluorescence microscope. Growth of GFP-RH was calculated using Image-Pro-Express.

Effect of Ov EO and Ca on the Invasion of T. gondii
Invasion experiments were performed as described by Augusto et al. (2018). HFF cells was cultured in a 6-well plate, and 3 ml DMEM with 2% FBS was added to each well. Then, 10 4 RH and 17 mg/ml Ov EO or Ca were added simultaneously to the wells, respectively, incubating for 20, 40, or 60 min. The supernatant was gently removed, cells were fixed with 2 ml methanol for 10 min, washed three times with PBS, blocked by 5% solution of BSA in PBS (BSA/PBS) for 1 h, and washed three times with PBS. This was then incubated with mouse anti-Toxoplasma SAG1 monoclonal antibodies (mAb), diluted (1:1,000) with a 1% BSA/PBS solution, at room temperature for 2 h. Then, goat anti-mouse IgG H&L(FITC) secondary antibodies, diluted (1:1,000) in 1% BSA/PBS, were added to 6-well plates and incubated at room temperature for 2 h. After washing thrice with PBS, 300 ml of 0.2% Triton X-100 was added, and the mixture was left for 30 min. Cells were then gently washed three times with PBS, and 300 ml of a 5% BSA/PBS solution was added dropwise for a second blocking. The antibodies were added as per the procedure described earlier, this time using goat antimouse IgG H&L (Alexa Fluor ® 568) (ab175473) instead of the goat anti-mouse IgG H&L (FITC). Finally, 300 ml of 30% glycerol was added to each well. Five visual fields were randomly selected for observation under the 40× objective of the fluorescence microscope and the parasites in each field were counted. Three repetitions were performed to increase the accuracy of the experiment. The difference between the tachyzoites of the two colors is termed as the absolute invasion number of tachyzoites. The ratio of the invasion number to the total number of tachyzoites is termed as the invasion rate of tachyzoites.

Assessment of Tachyzoite Ultrastructure Using Scanning Electron Microscopy
To determine differences in the ultrastructure of tachyzoites after treatment, the purified tachyzoites were treated with 17 mg/ml Ov EO or Ca, respectively. After being cultured at 37°C for 4 h, they were washed gently with PBS twice, and fixed overnight with 2.5% glutaraldehyde at room temperature. Gradient dehydration was carried out with 30, 50, 70, 80, 90, 95 and 100% ethanol respectively, and the critical point drying was carried out after dehydration. The tachyzoites were coated with gold (20−30 nm) and observed by scanning electron microscopy.

Statistical Analysis
All data were analyzed using GraphPad Prism 8.0. The antiparasitic activity of the Ov EO and Ca was analyzed using an unpaired t-test, while the cell invasion data were processed using multiple t-tests, to compare the results of the test groups and those of the control group (*P <0.05, **P <0.01, ***P <0.001).

Cytotoxicity of Ov EO and Ca
The cytotoxic potential of Ov EO on the host cell was confirmed before the antiparasitic activity study. According to MTT assay result, the concentration that induced 50% HFF cells mortality (CC 50 ) of Ov EO was 134.9 mg/ml ( Figure 1). After the antiparasitic activity of Ov EO was confirmed, the cytotoxic potential of Ca was carried out, and the result indicated that the CC 50 of Ca was 43.93 mg/ml (Figure 1).

Antiparasitic Activity of Ov EO and Ca In Vitro
The anti-T. gondii activity of Ov EO was preliminarily evaluated by plaque test. As seen in Figure 2A, we found that the plaques visible were fewer in number and smaller in size after treatment with 9 or 17 mg/ml Ov EO (Figures 2Aa, b), as compared to those in the DMSO-treated and untreated groups. These results indicated that Ov EO was able to inhibit the growth of RH within safe concentrations. In order to find the effective ingredients in Ov EO that have the role of anti-toxoplasma, Ca was evaluated by plaque test. The results indicated that Ca was able to inhibit the growth of RH at 9 or 17 mg/ml (Figures 2Ae, f). The results indicated that the growth of T. gondii was inhibited by each of the concentrations of Ov EO and Ca tested ( Figure 2B). To confirm and evaluate the effect of Ov EO concentration on anti-parasitic activity, five different concentrations were compared using an in vitro inhibition assay. The results indicated that the growth of T. gondii was inhibited by each of the concentrations of Ov EO tested (Figure 3), and the inhibition increased in a dose-dependent manner ( Figure 4A), the IC 50 of Ov EO was 16.08 mg/ml. We could find that the growth of T. gondii was significantly reduced after treatment with 70 and 35 mg/ml Ov EO (71 vs. 1,337; 320 vs. 1,337, P <0.001), as compared to the untreated and 1.56% DMSO-treated groups.
To compare the effect between Ov EO and Ca on antiparasitic activity, four different concentrations of Ca were compared using the same assay. The results indicated that the growth of T. gondii was inhibited by each of the concentrations of Ca tested (Figure 3), and the inhibition also increased in a dose-dependent manner ( Figure 4B), the IC 50 of Ca was 7.688 mg/ml. Comparing to the untreated group, the growth of T. gondii was also significantly reduced in low Ca concentrations treated groups (9 and 4 mg/ml) (606.8 vs 1,337; 980.7 vs. 1,337, P <0.05). Both Ov EO and Ca inhibited the growth of T. gondii in a dose-dependent manner, for the groups treated with 17 mg/ml Ov EO and 17 mg/ml Ca, the differences were also significant (692.9 vs. 1,337, P <0.01; 225.5vs. 1,337, P <0.001). For the inhibition effect, Ca was better than Ov EO at the same concentration. The inhibition of T. gondii was much more significant in the groups treated with 17 mg/ml Ca, than in those treated with SMZ (225.5 vs. 1,337, P <0.001; 490 vs. 1,337, P <0.01). There was no significant decrease in untreatment groups after treatment with 1.56% DMSO, which indicated that DMSO had no inhibitory effect on GFP-RH (1,322 vs 1,337). Due to the results of inhibition, we can calculate the IC 50 of Ov EO and Ca was 16.08 and 7.688 mg/ml, respectively ( Figure 5). After statistical analysis, the selectivity index (SI) of Ov EO and Ca was 8.389 and 5.714, respectively ( Table 1). From the comprehensive comparison of safety, the performance of Ov EO is better than that of Ca.

Effect of Ov EO and Ca on the Invasion of T. gondii
As summarized in Figure 6, in the 17 mg/ml Ov EO treatment group, the T. gondii invasion rates at 20, 40, and 60 min postinfection were found to be 17.84, 24.10, and 28.96% respectively ( Figure 6A). In the untreated group, invasion rates were found to be 38. 85, 47.52, and 54.70% respectively at the three time points ( Figure 5A). The invasion of T. gondii was significantly inhibited by Ov EO at 40 min (24.10% vs. 47.52%, P <0.01) and 60 min (28.96% vs. 54.70%, P <0.05). In the 17 mg/ml Ca treatment group, the T. gondii invasion rates were 21.09, 27.51, and 32.03% respectively at the three time points (Figure 6B), and the control group, invasion rates were found to be 30.52, 51.20, and 57.81% respectively ( Figure 6B). Compared to the untreated group.  Ca significantly reduced the invasion of T. gondii, especially after treatment for 40 and 60 min (27.51% vs. 51.20%, P <0.001; 32.03% vs. 57.81%, P <0.05, Figure 6B). The inhibitory effect was observed to increase as the treatment time increased. No change in the invasion rate of T. gondii was observed in any group treated with DMSO, across all experiments.

Tachyzoite Ultrastructure Analysis
The Scanning electron microscopy (SEM) results showed that the tachyzoites curled into a head-to-tail shape after the Ca treatment, but the individual was still plump and not shriveled ( Figure 7B). After treatment with Ov EO, the morphology of the tachyzoites changed significantly, the worms were distorted, and showed a certain shriveled state ( Figure 7A). In comparison, the tachyzoites were normal full crescent-shaped in DMSO treated group ( Figure 7C) and untreated group ( Figure 7D).

DISCUSSION
T. gondii is an important zoonotic parasite, which can cause serious consequences after infected different hosts (Elsheikha, 2008;Maldonado and Read, 2017;El Bissati et al., 2018). In the face of the current dilemma of treatment and prevention, it is urgent to explore new drugs to inhibit T. gondii and control toxoplasmosis. At present, several reports indicated that plant   7-81.92, 73.9, 63.03, and 30.73%, respectively (Leyva-Loṕez et al., 2017). This was the reason we chose the Ca study whether it plays an anti-Toxoplasma effect in Ov EO. The cytotoxicity of Ov EO is related to the extraction method, and its toxicity to different cell lines is also different. The cytotoxicity of methanolic extracted Ov EO was more toxic, the CC 50 of it was 382-374 mg/ml in MCF-7 cells. Chaouki reported that the CC 50 of Ov EO was 5.5, 5.2, and 7.5 mg/ml in breast cancer cells (MCF-7), lung cancer cells (H-460) and central nervous system cancer cells (SF-268), respectively (Chaouki et al., 2010). In our experiment, the CC 50 of Ov EO was 134.9 mg/mL in HFF. It has quite low cytotoxicity, so we can continue to carry out follow-up studies to evaluate its anti-Toxoplasma effect. The CC 50 of Ca was 43.93 mg/ml, which is more toxic than Ov EO in HFF cells. Mostly, the active ingredients are more toxic than essential oil mixtures, and the effect will be better. From our results we found that the SI of Ov EO and Ca was not very high, which means that the cytotoxic of them are quite high, so further study should be carry on to reduce their cytotoxicity. Gaur et al. (2018) also found that Ca was more toxic than Ov EO in HCT-8 cells. In our experiment, Ca showed better anti-T. gondii activity than Ov EO at same concentration. As a kind of phenol, Ca has a significant repellent effect on many kinds of insects, such as Aedes albopictus, Culex quinquefasciatus, Ixodes scapularis, Rhipicephalus appendiculatus and so on (Lima et al., 2019). Moreover, Ca also shows anti-nematode effect on different nematodes such as Ascaris suum (Trailovićet al., 2015). The main reason is that Ca can inhibit the contraction of muscle cells induced by acetylcholine, thereby inhibiting the muscle contraction of the parasite and affecting its motility (Marjanovicé t al., 2020). The above speculation is that the mechanism of carvacrol against T. gondii is related to the restriction of the motility of the parasite. However, its effect on the stability of calcium ions is also worthy of attention. Carvacrol has a certain regulatory effect on the stability of intracellular calcium ions. Studies have shown that carvacrol can adjust the TRP channels of transient calcium permeation receptor potentials, such as TRPV3, thereby increasing the concentration of calcium ions in the cytoplasm of primary mouse corneal epithelial cells and cultured human corneal epithelial cells (HCE-T cells) (Yamada et al., 2010). As we all know, there is a very important Ser/Thr protein kinase family in T. gondii, the CDPK family, whose activity is directly regulated by calcium ions. One family member, CDPK1, is closely related to the adhesion and invasion of T. gondii. Therefore, the reason why carvacrol can inhibit T. gondii may be achieved by inhibiting the activity of its invasion-related proteins. In our results, Ca did significantly inhibit the invasion rate of tachyzoites on HFF cells.
According to the results of SEM, the tachyzoites curled into a head-to-tail shape after the Ca treatment. Obviously, this obvious morphological change caused damage to the mobility of tachyzoites, which in turn affected its ability to invade. Studies have shown that carvacrol may induce apoptosis by reducing mitochondrial potential, releasing cytochrome C, activating caspase and carving PARP, thereby inhibiting human metastatic breast cancer cell line (MDA-MB 231) or human non-small cell lung cancer cell line Proliferation (A549) (Suntres et al., 2015). Therefore, the specific mechanism of Ca inhibiting T. gondii is not yet fully understood, and further research is needed. After Ov EO treatment, the tachyzoites showed severe dehydration and dryness. Ov EO is a mixture of different components, including terpenes, aldehydes, alcohols, esters, phenolic, ethers, and ketones and so on (Swamy et al., 2016). Among them, phenol can dehydrate the cells, resulting in the desiccation phenomenon (Samie et al., 2019). In addition, some FIGURE 7 | Scanning electron microscopy assay. The T. gondii were treated with 17 mg/ml Ov EO (A), 17 mg/ml Ca (B), DMSO (C) or untreated (D). After treated by Ov EO, the tachyzoites became sunken compared with those untreated tachyzoites. Tachyzoites treated with Ca become curled up. Scale bars: 1 mm.

A B
FIGURE 6 | Effect of Ov EO on the invasion of T. gondii. Statistics of T. gondii invasion rate using two immunofluorescent dyes after treated with Ov EO (A) and Ca (B) for 20, 40, 60 min, respectively. *P < 0.05, **P < 0.01, ***P < 0.001 compared with untreated group.
hydrophobic compounds can pass through biological barriers and biological membranes, which may also have anti-T. gondii effects (Costa et al., 2018).

CONCLUSION
In this study, we found that Ov EO and Ca had obvious anti-Toxoplasma effect, which is likely to be achieved by changing the shape of tachyzoites, thereby limiting its movement ability, and then affecting its invasion ability. At the same time, Ca may have other biological functions, which can inhibit the proliferation of T. gondii. However, the target molecular and mechanism of action of Ov EO on T. gondii are still unclear and warrant further studies.

DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.