Ethanol Extracts of Achillea millefolium and Hypericum perforatum Low Anti-Toxoplasma Activity.

OBJECTIVES: This study was performed to determine the lethal and the inhibitory effects of ethanol extracts of Achillea millefolium (A. millefolium) and Hypericum perforatum (H. perforatum) on Toxoplasma gondii (T. gondii) RH strain tachyzoites in vitro.
METHODS: The tachyzoites were treated with concentrations of 10, 50, and 100 mg/mL of A. millefolium and H. perforatum extracts within 10, 30, and 45 minutes in the wells. The mortality rates of tachyzoites treated with extracts were determined by using alkaline methylene blue staining. Also, the tachyzoites in cell cultures were treated with concentrations of 50, 100, and 200 mg/mL of these extracts. The cell viability, inhibition concentration (IC50), and selectivity were determined from MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays.
RESULTS: In the cell-free in vitro study, all of tachyzoites were killed at concentrations of 100 mg/mL of both extracts while at concentration 10 mg/mL, the mortality was 4.53% - 5.31%. In the cell culture study, the values of the effective concentration (EC50) were 215 and 153 μg/mL and the selectivities were 0.73 and 0.69 for the A. millefolium and the H. perforatum extracts, respectively.
CONCLUSION: We conclude that neither extracts has any significant effect on the tachyzoites of T. gondii in cell cultures.

1. Introduction

Toxoplasma gondii (T. gondii) is one of the most common parasitic zoonosis. It can be found worldwide [1]. Treatment of toxoplasmosis is commonly performed with the synergistic drugs pyrimethamine and sulfadiazine. Although, synthetic drugs have acceptable anti-Toxoplasma activities, their adverse effects are limiting factors, especially in pregnant women with acute toxoplasmosis. For example, pyrimethamine causes suppression of hematopoiesis in patients [2]. Therefore, production of an effective anti-Toxoplasma drug with low side effects is a priority in Toxoplasma research.

Based on a PubMed search, some in vitro studies have done on the effectiveness of herbal products in treating T. gondii [3-5]. However, only one report, a report from Iran, was found on the anti-Toxoplasma effect of herbal extracts; that research studied the effect of garlic extract on acute toxoplasmosis in mice [6]. This study was performed in order to determine the lethal and the inhibitory effects of ethanol extracts of Achillea millefolium (A. millefolium) and Hypericum perforatum (H. perforatum) on T. gondii RH strain tachyzoites in vitro.

2. Materials and Methods

A. millefolium and H. perforatum were obtained from herbal marketing Jahad Daneshgahi Karaj (Tehran, Iran) and were confirmed by a botanist. Voucher specimens of the herbal plants were saved at the Herbarium Center of Medicinal Plants of the Academic Center for Education, Culture and Research (ACECR), Karaj, Iran. Aerial parts of plants were dried at room temperature and were powdered. Fifty grams of each herb were extracted by using the percolation method with 80% ethanol at room temperature. The samples were stored at 4ºC until use (3 gr, 6% yield). The dried plant extracts were dissolved in 1% dimethyl sulfoxide (DMSO) and diluted with phosphate buffer solution (PBS) to different concentrations. Also, pyrimethamine (Sigma, USA) dissolved in methanol-acetone (50% v/v) and diluted with Roswell Park Memorial Institute (RPMI) 1640 medium was used as a positive control.

We used T. gondii RH strain tachyzoites. HeLa cells were cultured and used to assay the anti-Toxoplasma effects of the herbal extracts. The assays were performed by treating a 50-μL tachyzoites suspensions containing nearly 5 × 105 tachyzoites with 50 μL of different concentrations of the extracts (10, 50, and 100 mg/mL) within 10, 30, and 45 minutes at room temperature. All treatments were assayed in triplicate.

The tachyzoites treated with both extracts that showed 100% mortality based on methylene-blue staining were bioassayed in mice. Fifty microliters of suspensions containing nearly 5 × 105 tachyzoites were inoculated intraperitoneally into 3 mice. All of the mice were monitored for up to one month after inoculation in terms of activity and mortality.

HeLa cell suspensions were cultured and within 24 hours after incubation, 100 μL of a suspension containing 3 × 105 fresh tachyzoites were added to each well. Six hours after the inoculation of the tachyzoites to wells, the cultures were washed twice with RPMI 1640 medium without fetal bovine serum (FBS) in order to remove non-adherent tachyzoites. Eighteen hours after incubation, the herbal extracts were individually added to the wells at a concentration of 50, 100, or 200 μg/mL. Twenty four hours after the addition of the herbal extracts, the anti-Toxoplasma activity and the cytotoxicity of those traditional medicines were examined using MTT (3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide) assay kits (Bio Idea Company, Tehran, Iran). Optical densities (ODs) were read using an enzyme-linked immunosorbent assay (ELISA) microplate reader (Epoch, USA) at a wavelength of 570 nm. All experiments were performed in triplicate. The results were expressed as percent cell viability, half maximal effective concentration (EC50), and selectivity.

3. Results

Both herbal extracts showed toxoplasmacidal effects. The results for the mortality rates (%) of the tachyzoites are shown in (Table 1). The mortality rate at a 100-mg/mL concentration of A. millefolium was significantly higher than that at a 50-mg/mL concentration (P < 0.001), but the difference in the mortality rates between of 10- and 50-mg/ mL concentrations of the extract were not significant. The toxoplasmacidal effects of the H. perforatum extract were similar at concentrations of 50 and 100 mg/mL; however, compared to the concentration of 10 mg/mL, both the 50- and the 100-mg/mL concentrations of the extract showed significant increases in those effects. Also, a 100% mortality rate of the tachyzoites in mice treated with the extracts was confirmed by using bioassays, and all of the mice inoculated with the tachyzoites were alive and active one month after the inoculation. Both herbal extracts showed anti-Toxoplasma activity in the cell culture; however, the EC50 of H. perforatum extract (153 μg/mL) was lower than the EC50 of A. millefolium extract (215 μg/mL), (Table 2).

Table. 1

Mortality (%) of T. gondii RH strain tachyzoites after treatment with ethanol extracts of H. perforatum and A. millefolium

Incubation time	Herbal extract type	Concentration (mg/mL)
(minutes)		10	50	100
10	H. perforatum	5.01 ± 1.09	100	100
	A. millefolium	4.53 ± 0. 91	5.97 ± 2.43	100
30	H. perforatum	5.22 ± 0.77	100	100
	A. millefolium	4.63 ± 0.75	6.11 ± 2.17	100
45	H. perforatum	5.31 ± 0.89	100	100
	A. millefolium	5.11 ± 1.12	6.26 ± 2.70	100

T. gondii, Toxoplasma gondii; A. millefolium, Achillea millefolium; H. perforatum, Hypericum perforatum.

Table. 2

In vitro anti-Toxoplasma activity and selectivity of A. millefolium and H. perforatum extracts and pyrimethamine

Herbal extract/drug	EC50
(μg/mL)	HeLa	HeLa+T. gondii	Selectivity*
A. millefolium	158	215	0.73
H. perforatum	105.9	153	0.69
Pyrimethamine	0.6	0.176	3.40

*Ratio of the EC50 value for HeLa cells to the EC50 value for T. gondii RH strain. A. millefolium, Achillea millefolium; H. perforatum, Hypericum perforatum; EC50, effective concentration; T. gondii, Toxoplasma gondii.

After T. gondii-infected HeLa cells had been incubated with different concentrations of the extracts, their viability decreased in a dose-dependent manner (Figs. 1,2). The viability showed significant decreases, compared with the control, at all concentrations of the extracts (P < 0.05). On the other hand, the inhibitory effect of pyrimethamine on cell proliferation was significantly higher than the inhibitory effects of the two herbal extracts (Figs. 1,2).

Fig. 1

Inhibitory effect of A. millefolium extract on T. gondii RH strain tachyzoites in cell cultures.

Fig. 2

Inhibitory effect of H. perforatum extract on T. gondii RH strain tachyzoites in cell cultures.

4. Discussion

In the present in vitro study, ethanol extracts of A. millefolium and H. perforatum showed toxoplasmacidal activities in RH strain tachyzoites exposed to those extracts and had inhibitory effects on the parasite in cell cultures. However, the anti-Toxoplasma activity of the H. perforatum extract was stronger than that of the A. millefolium extract, but their selective toxicities were low. Other studies on herbal extracts have also shown that Glycyrrhiza glabra L., Acorus gramineus Soland and Dryopteris crassirhizoma have anti- Toxoplasma activities [7, 8].

Previous studies have been shown that some extracts/fractions of medicinal plants have remarkable anti-Toxoplasma activities. The extracts of Sophora flavescens Aiton and Zingiber officinale have high anti-Toxoplasma activities (EC50 = 0.20 and 0.18) with high selectivities (selectivity = 4.6% and 10.1%, respectively) [8]. Furthermore, in cell cultures, the inhibitory effects of Torilis japonica and Sophora flavescens on T. gondii and Neospora caninum tachyzoites have been shown to be higher than those of Pulsatilla koreana, Ulmus macrocarpa, and Sinomenium acutum [3]. Furthermore, the study of Youn et al [4] also showed that some high-performance liquid chromatography (HPLC) fractions of those plants have more efficient on tachyzoites in cell cultures [4].

5. Conclusion

Our study showed that the anti-Toxoplasma activities of A. millefolium and H. perforatum extracts were significantly lower than that of pyrimethamine which is a common synthetic drug for the treatment of toxoplasmosis. Therefore, these plants do not seem to be good candidates for continuance of anti-Toxoplasma studies. However, further studies to clarify the anti-Toxoplasma activities of other herbs are recommended.

*P < 0.01, compared to pyrimethamine and †P < 0.001 compared to control (Tukey Kramer test). A. millefolium, Achillea millefolium; T. gondii, Toxoplasma gondii; C, control; Pyr, pyrimethamine; Achi, Achillea millefolium.

*P < 0.01 compared to pyrimethamine and †P < 0.001 compared to control (Tukey Kramer test). H. perforatum, Hypericum perforatum; T. gondii, Toxoplasma gondii; C, control; Pyr, pyrimethamine; Hyper, Hypericum perforatum.