In vitro and in vivo Anti- Toxoplasma Effects of Allium sativum Essential Oil Against Toxoplasma gondii RH Strain

Abstract

Background: Since no effective vaccine has been developed for toxoplasmosis, prophylaxis in seronegative pregnant women and immunocompromised patients with a CD4 <100 cells/μL is highly recommended as an ideal strategy to prevent this disease. This study aimed to assess the chemical composition, in vitro, and in vivo effects of Allium sativum essential oil (ASEO) against Toxoplasma gondii RH strain.

Methods: The in vitro anti-Toxoplasma effects of different concentrations of ASEO (32.5, 75, 150 µg/mL) were measured by MTT assay for 0.5, 1, 2, and 3 h. Male Balb/c mice were orally administrated ASEO at the doses of 200, 400, and 600 µg/kg/day for 14 days. One day after the completion of oral drug administration, the mice in all groups were infected intraperitoneally with 1×10⁴ tachyzoites. They were checked daily and the rate of survival was recorded. The peritoneal fluids of the mice were collected and the mean number of tachyzoites was calculated via a light microscope. The level of liver lipid peroxidation (LPO) and nitric oxide (NO), toxicity effects on the liver and kidney, and the mRNA expression levels of some pro-inflammatory cytokines such as IL-1β and IFN-γ were determined by quantitative real-time PCR.

Results: Different concentrations of ASEO showed a significant (p < 0.001) anti-Toxoplasma activity against T. gondii tachyzoites, and the highest efficacy was observed at the concentration of 150 µg/mL. Fourteen days of pre-treatment of infected mice with ASEO at the doses of 200, 400, and 600 µg/kg/day significantly (p < 0.001) decreased the mean number of tachyzoites and mortality rate by the 6th, 7th, and 8th days after infection, respectively. ASEO at the doses of 200, 400, and 600 µg/kg/day significantly (p < 0.05) improved the increase in the LPO and NO. Pre-treatment of mice with different doses of ASEO provoked a considerable (P < 0.001) downregulation of IL-1β and IFN-γ mRNA gene expression levels, but it had no significant toxicity on the serum levels of some liver and kidney enzymes.

Conclusion: The present study demonstrated the considerable prophylactic effects of ASEO that increased the survival rate of mice and reduced the parasite load in them. Our findings also showed that ASEO promotes the innate immune system, pro-inflammatory cytokines, inhibition of hepatic injury, etc. in the mice with acute toxoplasmosis. However, additional investigations are mandatory to clarify the accurate prophylactic and therapeutic anti-Toxoplasma mechanisms of ASEO as well as all its toxicity aspects, especially in clinical settings.

Keywords: RH strain; herbal medicines; prophylaxis; tachyzoites; toxoplasmosis.

Figure 1

In vitro anti-Toxoplasma effects of different concentration of ASEO showed against T. gondii tachyzoites after 0.5, 1, 2, and 3 h incubation in comparison with the control group. Data are expressed as the mean ± SD (n = 3).

Figure 2

The infection rate of the Vero cells infected with T. gondii tachyzoites after exposed with Allium sativum essential oil (ASEO) at concentrations of 32.5, 75, 150 µg/ml for 3 h (A). The intracellular replication of T. gondii in infected Vero cells after treatment with various concentrations of ASEO (B).Data are expressed as the mean ± SD (n = 3). * p<0.05.

Figure 3

The mortality rate infected mice pre-treated with ASEO at the doses of 200, 400, and 600 µg/kg/day for 14 days in comparison with the control group. Data are expressed as the mean ± SD (n = 8).

Figure 4

The mean number of tachyzoites in the infected mice pre-treated with ASEO at the doses of 200, 400, and 600 µg/kg/day for 14 days. Data are expressed as the mean ± SD (n = 8). *p < 0.05; **p < 0.001.

Figure 5

The level of hepatic MDA and NO in the T. gondii infected mice pre-treated ASEO at the doses of 200, 400, and 600 µg/kg/day. Data are expressed as the mean ± SD (n = 8). *p < 0.05; **p < 0.001.

Figure 6

The expression level of IFN-γ and IL-1β mRNA in the T. gondii infected mice pre-treated ASEO at the doses of 200, 400, and 600 µg/kg/day. Data are expressed as the mean ± SD (n = 8). *p < 0.05; **p < 0.001.

Figure 7

The level of biochemical factors in mice sera after oral administration of ASEO for 14 days. Data are expressed as the mean ± SD (n = 8).