Evaluation of Anti-Toxoplasma gondii Effect of Ursolic Acid as a Novel Toxoplasmosis Inhibitor

This study was carried out to evaluate the anti-parasitic effect of ursolic acid against Toxoplasma gondii (T. gondii) that induces toxoplasmosis, particularly in humans. The anti-parasitic effects of ursolic acid against T. gondii-infected cells and T. gondii were evaluated through different specific assays, including immunofluorescence staining and animal testing. Ursolic acid effectively inhibited the proliferation of T. gondii when compared with sulfadiazine, and consistently induced anti-T. gondii activity/effect. In particular, the formation of parasitophorous vacuole membrane (PVM) in host cells was markedly decreased after treating ursolic acid, which was effectively suppressed. Moreover, the survival rate of T. gondii was strongly inhibited in T. gondii group treated with ursolic acid, and then 50% inhibitory concentration (IC50) against T. gondii was measured as 94.62 μg/mL. The T. gondii-infected mice treated with ursolic acid indicated the same survival rates and activity as the normal group. These results demonstrate that ursolic acid causes anti-T. gondii action and effect by strongly blocking the proliferation of T. gondii through the direct and the selective T. gondii-inhibitory ability as well as increases the survival of T. gondii-infected mice. This study shows that ursolic acid has the potential to be used as a promising anti-T. gondii candidate substance for developing effective anti-parasitic drugs.


Introduction
Toxoplasmosis is caused in all age groups, including young children or adults globally, which is one of parasitic diseases as zoonosis infected through Toxoplasma gondii (T. gondii). T. gondii not only induces chronic infection in various sites of human body, but it also causes brain infection through the central nervous system as zoonotic parasitosis. In addition, T. gondii has unique infectious subcellular organelles, including apicoplast, conoid, rhoptries, dense granules, and micronemes [1]. In general, T. gondii forms a unique proliferative membrane, such as a parasitophorous vacuole membrane (PVM) including reticular network (RN) during its proliferation after invasion into host cells, and grows in it [2]. Until recently, several drugs were developed to inhibit T. gondii, and they are being used to treat toxoplasmosis' patients in the clinic. However, their side effects are still clear in the clinic, and it is also being exposed to drug-resistance gradually [3][4][5]. In these aspects, pyrimethamine used for treating toxoplasmosis blocks the synthesis of tetrahydrofolic acid from dihydrofolate reductase (DHFR) by effectively inhibiting the dihydrofolate reductase in T. gondii, which inhibits consistently synthesis of the DNA and/or RNA in the proliferation of protozoa species, including malaria.
Until recently, it was reported that various extracts and/or natural products derived from medicinal plants had the possibility of useful medical resources for treating infectious virus diseases,

Effect of Ursolic Acid on the Proliferation and Growth of T. gondii
T. gondii causes parasitic disease on both animals and human, as well as having unique micro network systems and a specific structure, including various micro-organelles, such as mitochondria, rhoptries, and micronemes. We evaluated the anti-parasitic activity and the effects of UA on the proliferation and growth of T. gondii using an MTT assay and UA showed the anti-T. gondii activity in the range of 25-200 µg/mL. T. gondii-infected cells were incubated with different concentrations  µg/mL) of UA for 24 h, and their viability was markedly decreased in a dose dependent manner (Tables 1 and 2). In addition, the UA effectively inhibited the proliferation and growth of T. gondii-infected cells as compared with SF. In particular, T. gondi-infected cells treated with UA (100 µg/mL) showed a significant decrease of T. gondii including T. gondii fragmentation as well as morphological changes such as cell shrinkage and cell fragmentation when compared with the untreated T. gondii-infected cells (Data not shown), which suggests that UA has anti-parasitic activity and the inhibitory effect against T. gondi in infected cells (Table 2). Furthermore, the UA effectively inhibited the viability of the parasite through the direct inhibition of T. gondii when compared with SF, which is to strongly demonstrate its selective inhibitory effect against T. gondii, and then the parasitic SR was measured at less than 45% at concentrations of 100 µg/mL ( Figure 1). The 50% inhibitory concentration (IC 50 ) value of UA against T. gondii and T. gondii-infected cells was measured as 94.62 µg/mL and 162.25 µg/mL, respectively. These results demonstrate that UA strongly induced anti-proliferation activity of T. gondii in the infected host cells by effectively blocking T. gondii as well as the direct inhibitory action against T. gondii. The results were expressed as a percentage of the control group, and all of the results were presented as mean ± standard deviation (S.D.) of three independent experiments. * p < 0.05 was considered to be statistically significant.  The results were expressed as a percentage of the control group, and all of the results were presented as mean ± standard deviation (S.D.) of three independent experiments. * p < 0.05 was considered to be statistically significant.  The results were expressed as a percentage of the control group, and all of the results were presented as mean ± standard deviation (S.D.) of three independent experiments. * p < 0.05 was considered to be statistically significant. The anti-T. gondii effect of ursolic acid against the proliferation and the viability of T. gondii and T. gondii-infected cells was measured using the MTT assay. They were incubated with different concentrations (25-200 μg/mL) of the compound at 37 °C for 24 h respectively. IC50 (50% inhibitory concentration value of UA against the survival rate of T. gondii and T. gondii-infected cells). The results were carried out three times independently.

162.25
The anti-T. gondii effect of ursolic acid against the proliferation and the viability of T. gondii and T. gondii-infected cells was measured using the MTT assay. They were incubated with different concentrations (25-200 µg/mL) of the compound at 37 • C for 24 h respectively. IC 50 (50% inhibitory concentration value of UA against the survival rate of T. gondii and T. gondii-infected cells). The results were carried out three times independently. T. gondii-infected cells were incubated with different concentrations (25-200 µg/mL) of ursolic acid (UA) and sulfadiazine (SF) for 24 h respectively, and their survival rates were measured using the MTT assay. The results were expressed as a percentage of the normal group, and all the results were presented as mean ± standard deviation (S.D.) of three independent experiments.

Anti-Parasitic Effect of Ursolic Acid on PVM Formed by T. gondii Proliferation
T. gondii induces anti-apoptotic steps and features through the inactivation of apoptotic proteins during its proliferative phase in host cells, which shows the unique parasitic life-cycle of T. gondii. In particular, the formation of PVM is accelerated in a time-dependent manner after cell invasion of T. gondii. For this reason, we investigated the inhibition of PVM that is caused by the interaction between T. gondii and UA during the proliferation stage of T. gondii in host cells. As shown in Figure 2, the PVM formation and nucleus of T. gondii were markedly decreased in T. gondii-infected host cells when it was treated with 100 µg/mL of UA and SF, respectively, and their changes were clearly observed under a UV fluorescence. The results indicate the inhibitory effect of UA against PVM formation and the viability of T. gondii. Therefore, these results show substantial evidence that UA effectively inhibited or blocked the PVM formed by T. gondii during the proliferative stage as well as the proliferation of T. gondii in the infected host cells after cell invasion. T. gondii-infected cells were incubated with different concentrations (25-200 μg/mL) of ursolic acid (UA) and sulfadiazine (SF) for 24 h respectively, and their survival rates were measured using the MTT assay. The results were expressed as a percentage of the normal group, and all the results were presented as mean ± standard deviation (S.D.) of three independent experiments.

Anti-Parasitic Effect of Ursolic Acid on PVM Formed by T. gondii Proliferation
T. gondii induces anti-apoptotic steps and features through the inactivation of apoptotic proteins during its proliferative phase in host cells, which shows the unique parasitic life-cycle of T. gondii. In particular, the formation of PVM is accelerated in a time-dependent manner after cell invasion of T. gondii. For this reason, we investigated the inhibition of PVM that is caused by the interaction between T. gondii and UA during the proliferation stage of T. gondii in host cells. As shown in Figure 2, the PVM formation and nucleus of T. gondii were markedly decreased in T. gondii-infected host cells when it was treated with 100 μg/mL of UA and SF, respectively, and their changes were clearly observed under a UV fluorescence. The results indicate the inhibitory effect of UA against PVM formation and the viability of T. gondii. Therefore, these results show substantial evidence that UA effectively inhibited or blocked the PVM formed by T. gondii during the proliferative stage as well as the proliferation of T. gondii in the infected host cells after cell invasion.

Effect of Ursolic Acid through the Inhibition of T. gondii in T. gondii-Infected Mice
T. gondii induces infectious symptoms and diseases, such as lymphadenopathy and brain injury, particularly in human as a zoonotic parasite that is infected in both human and animals. In this aspect, we evaluated the anti-proliferative effect and the parasitic inhibitory activity of UA against the viability of T. gondii in T. gondii-infected mice. The mice were carefully observed during the experimental periods after the injection of T. gondii treated with 100, 200, and 400 μg/mL of UA for 24 h. As mentioned above, T. gondii-infected mice treated with different concentrations (200 and 400 μg/mL) of UA showed higher activity and viability than T. gondii-infected mice, and the mice indicated the dynamic and energetic activity as the normal group. In particular, it clearly confirmed  T. gondii induces infectious symptoms and diseases, such as lymphadenopathy and brain injury, particularly in human as a zoonotic parasite that is infected in both human and animals. In this aspect, we evaluated the anti-proliferative effect and the parasitic inhibitory activity of UA against the viability of T. gondii in T. gondii-infected mice. The mice were carefully observed during the experimental periods after the injection of T. gondii treated with 100, 200, and 400 µg/mL of UA for 24 h. As mentioned above, T. gondii-infected mice treated with different concentrations (200 and 400 µg/mL) of UA showed higher activity and viability than T. gondii-infected mice, and the mice indicated the dynamic and energetic activity as the normal group. In particular, it clearly confirmed a significant difference of the survival rate between T. gondii-infected mice and the infected mice treated with UA. However, T. gondii-infected mice treated with 100 µg/mL of UA showed that the effect of UA did not consistently maintain the survival of the infected mice by decreasing after 11.8 days. This result suggests that the death of mice may be owing to tissue damage and cytolysis by invasion of T. gondii than the loss of body weight of mice. Furthermore, symptoms and side effects, such as the loss of body weight, were not observed or induced in all of the groups treated with 200 and 400 µg/mL of UA for the experimental periods ( Figure 3). The result shows effective and clear evidence regarding the effect of anti-T. gondii compounds, which indicates clarity regarding the results as a fusion method for both "in vitro" and "in vivo". These results demonstrate that UA has the selective anti-parasitic activity which strongly causes anti-T. gondii effects as well as suppressing their viability by effectively inhibiting T. gondii.  The mice were carefully observed during the experiment periods after T. gondii infection. The results were expressed as a percentage of the normal group, and presented as the mean ± S.D. * p < 0.05 was considered to be statistically significant.

Discussion
Until recently, the efforts for the treatment of neglected infectious diseases have been attempted at various fields. In particular, the global supporting organizations, including WHO, Bill & Melinda Gates Foundation, and the global pharmaceuticals are supporting the study for developing novel drugs and for effectively treating or blocking zoonosis such as malaria and tuberculosis, as well as infectious diseases, including AIDS, Zika, Ebola, MERS, and SARS. We should carefully focus on the neglected infectious diseases that are consistently caused through various pathways and infection factors. From these perspectives, T. gondii causes to fetus serious infectious diseases such as retinochoroiditis, hydrocephalus, and cerebral calcification through fetus infection during pregnancy, as well as induces symptoms, such as lymphadenopathy and meningoencephalitis in brain through acquired infection as a zoonotic parasite that is infected in both human and animals. It also induces serious complications in immune deficient patients and HIV patients as one of the infectious parasites that induce parasitic zoonosis, which may induce the interaction for parasitic The mice were carefully observed during the experiment periods after T. gondii infection. The results were expressed as a percentage of the normal group, and presented as the mean ± S.D. * p < 0.05 was considered to be statistically significant.

Discussion
Until recently, the efforts for the treatment of neglected infectious diseases have been attempted at various fields. In particular, the global supporting organizations, including WHO, Bill & Melinda Gates Foundation, and the global pharmaceuticals are supporting the study for developing novel drugs and for effectively treating or blocking zoonosis such as malaria and tuberculosis, as well as infectious diseases, including AIDS, Zika, Ebola, MERS, and SARS. We should carefully focus on the neglected infectious diseases that are consistently caused through various pathways and infection factors. From these perspectives, T. gondii causes to fetus serious infectious diseases such as retinochoroiditis, hydrocephalus, and cerebral calcification through fetus infection during pregnancy, as well as induces symptoms, such as lymphadenopathy and meningoencephalitis in brain through acquired infection as a zoonotic parasite that is infected in both human and animals. It also induces serious complications in immune deficient patients and HIV patients as one of the infectious parasites that induce parasitic zoonosis, which may induce the interaction for parasitic proliferation and growth through parasitic relationship such as symbiosis in the host. In this aspect, T. gondii not only inhibits defensive mechanisms of cytokines, such as IL-2, -4, -6, -8, and IFN-γ released from host cells after invasion, but also suppresses the production of protective systems that are activated through the immune-response. Furthermore, it is known to block the signaling transport pathways that activate anti-parasitic system and the resistance to T. gondii invasion in host cells, as well as to suppress the apoptotic signals and cell arrest pathways during the early apoptotic stage of host cells after cell invasion [32,33]. Moreover, T. gondii promotes inactivation of cell cycle initiators and apoptotic mediators during the proliferative phase of T. gondii by forming PVM of T. gondii in host cells, which rapidly accelerates the proliferation of T. gondii in PVM [34].
In this study, we evaluated the anti-parasitic activity/effect of UA which inhibits the proliferation of T. gondii, and confirmed the viability of T. gondii after the parasite infection through T. gondii-infected in vitro system and animal testing. In the present study, the expression of PVM in the infected host cells was significantly increased when compared with T. gondii-infected cells treated with UA and SF, and uninfected cells. On the other hand, the proliferation of T. gondii and PVM were markedly inhibited in T. gondii-infected cells treated with UA compared with T. gondii-infected cells. In addition, we evaluated the anti-T. gondii activity of UA through T. gondii-infected mice. After the injection of T. gondii treated with UA (200 and 400 µg/mL), the mice showed the vitality and the viability such as the uninfected control group without the loss of body weight and the cytotoxicity during the experiment. In particular, it was recently reported that UA has the anti-protozoa effects against Leishmania amazonesis and Leishmania infantums, causing leishmaniasis among the neglected tropical diseases, and its mechanism of action is associated with programmed cell death and nitric oxide (NO) production [35,36]. In this aspect, the anti-T. gondii effect of UA suggests that UA may induce the anti-proliferation and the growth inhibitory action of T. gondii through the mechanism of action that is caused by nitric oxide (NO) production or programmed cell death in T. gondii and the infected host cells.
Taken together, these results indicate that the expression of PVM in T. gondii-infected cells was selectively inhibited or blocked in the host cells treated with UA, as well as significantly increased in infected cells. Furthermore, the T. gondii-infected mice treated with UA showed a stable life-cycle and survival rates when compared with infected positive group during the experimental period. The results clearly provide the inhibitory effect and activity of the compound against T. gondii, and indicate whether UA induces the direct inhibition of T. gondii or increase the survival of mice by effectively blocking and inhibiting the viability of T. gondii.

Animals
BALB-c/mice (six weeks, n = 25) were purchased from DaeHan Bio-Link Co., Ltd. (Chungcheongbuk-do, Korea), and all animals were kept at 23 ± 0.5 • C and 12 h-light/dark cycle in a controlled environment of a central animal care facility. Food and water were provided ad libitum to all animals. The facility was strictly maintained in accordance with the guidelines of the National Institutes of Health for the Care and Use of Laboratory Animals.

Preparation of Anti-T. gondii Drugs
The anti-T. gondii drug, sulfadiazine (SF), was dissolved in DMSO and ursolic acid (UA) was also dissolved in DMSO to a concentration of 50 mg/mL, according to the manufacturer's instruction. Sulfadiazine was used as a standard drug to evaluate whether or not ursolic acid has an anti-parasitic effect and activity against T. gondii. All of the compounds were filtered using 0.2 µm membrane syringe filters (Roshi Kaisha, Ltd., Tokyo, Japan) before use, and were stored at −80 • C deep-freezer until use.

Cell Lines and Culture Conditions of T. gondii
Glioma cells (C6 cells) were purchased from Korean Cell Line Bank at Seoul National University. C6 cells were cultured in RPMI medium 1640 containing 2 mM L-glutamine, supplemented with 10% fetal bovine serum (FBS), 100 units/mL penicillin, and 100 µg/mL streptomycin (Biofluids, Rockville, MD, USA) in a humidified atmosphere containing 5% CO 2 in air at 37 • C. The RH strain of T. gondii was suspended with 1X PBS, which was injected in the abdominal cavity of each BALB-c/mouse. Five days after the injection, T. gondii was collected from the peritoneal fluids of each mouse kept in the abdominal cavities of the mice before use in the studies. In the in vitro study, cells were infected with T. gondii (cells:T. gondii = 1:5).

Evaluation of The Viability of T. gondii
To evaluate the inhibitory effects of UA against the viability of T. gondii, we investigated the viability of T. gondii exposed to UA and SF. Briefly, after T. gondii was seeded in a 24 well plate (1 × 10 7 /well), T. gondii was incubated with different concentrations (50-200 µg/mL) of UA and SF for 24 h, respectively, and their viabilities were determined by MTT assay. It is to determine whether or not UA has direct anti-parasitic activity and/or effect against T. gondii. The survival rate (SR) of T. gondii was calculated, as follows: % of SR = (OD drug-tested wells − OD blank )/(OD control − OD blank ) × 100. The optical density (OD) was measured at a wavelength of 570 nm using an ELISA leader.

Microscopic Observation of T. gondii in Infected Cells
The cells were seeded in a 24 well plate (1 × 10 5 /well), which were incubated at 37 • C for 24 h. The cells were infected with T. gondii (5 × 10 5 tachyzoites/well), and then T. gondii-infected cells were treated with 100 µg/mL of UA and SF for 24 h, respectively. The morphological changes of T. gondii-infected host cells were observed under a light microscope (Nikon Eclipse TE 2000-U, Tokyo, Japan).

Nuclear Staining of T. gondii-Infected Host Cells
The anti-T. gondii effect of UA against T. gondii-infected cells was evaluated using Hoechst 33342, according to the nuclear staining method described previously [37]. The cells were seeded onto cover slips in a 24 well plate (1 × 10 5 cells/well), which were infected with T. gondii (5 × 10 5 tachyzoites/well) after 24 h. The T. gondii-infected cells were incubated with 100 µg/mL of UA and SF for 24 h respectively. After washing with 1X PBS, the T. gondii-infected cells were fixed in 1X PBS containing 5% formaldehyde for 30  staining, the cells were washed with 1X PBS three times, and their nuclear changes were observed under a UV fluorescent microscope (Nikon Eclipse TE 2000-U, Tokyo, Japan).

PVM Formation in T. gondii-Infected Cells
The cells were seeded onto cover slips in a 24 well plate (1 × 10 5 cells/well), which were infected with T. gondii (5 × 10 5 tachyzoites/well). T. gondii-infected cells were incubated with 100 µg/mL of UA and SF for 24 h, respectively. After washing with 1X PBS, they were fixed with 5% formaldehyde for 10 min and 0.05% (v/v) Triton X-100 for 5 min. The cells were blocked with 1X PBS containing 1% BSA for 1 h at room temperature after washing. A mouse monoclonal anti-PVM antibody was diluted with 1:100 (v/v) using 1% BSA/PBS, and then the cells were incubated with anti-PVM antibody solution at room temperature for 1 h. After washing, goat anti-mouse IgG-FITC-conjugated secondary antibody was diluted with 1:100 (v/v) using 1X PBS, which was added to each well. The cells were incubated at room temperature for 1 h, and were washed with 1X PBS every 15 min, four times. Their fluorescence was observed under a UV fluorescent microscope (Nikon Eclipse TE 2000-U, Tokyo, Japan).

The Survival Rate of T. gondii-Infected Mice
Twenty-five animals (mouse/6 weeks, n = 25) were divided into normal (n = 5) and experimental groups (four groups, n = 20). T. gondii was seeded in a 12 well plate (4 × 10 6 /well), which was incubated with different concentrations (100, 200, and 400 µg/mL) of UA for 24 h, respectively. T. gondii treated with the compounds was harvested before the injection in the abdominal cavity of each mouse in the experiment group, which was washed with 1X PBS three times. The pellets were suspended with 1X PBS, which were injected in the abdominal cavity of each BALB-c/mouse in the experimental groups. T. gondii-infected mice untreated with UA were used as an infected positive group, and the animals were kept in a central animal care facility during the experiment.

Statistical Analysis
All of the results were expressed as mean ± S.D. Statistical analysis of the data was performed using Student's t-test and analysis of variance (ANOVA). * p < 0.05 was considered to be statistically significant.

Conclusions
The results of this study demonstrate that UA not only has anti-T. gondii activity causing the direct inhibition of T. gondii, but also causes anti-parasitic effect against T. gondii by strongly inhibiting the proliferation and growth of T. gondii in infected host cells. In addition, our results show clearly for the first time that UA has anti-T. gondii effect which consistently induces the survival of T. gondii-infected mice. Therefore, this study provides substantial results and the potential that UA can be utilized as a promising candidate substance for anti-T. gondii drug development of next-generation through T. gondii-infected cells and infected-mice model. In addition, UA suggests novel perspectives of the approach for anti-T. gondii drug development, which shows the need of further study regarding the safety and/or efficacy against toxoplasmosis through preclinical study in the near future.