Introduction

Despite great scientific advances of humans, giardiasis is still one of the most common parasitic diseases producing diarrhea in children worldwide. In 1998, the World Health Organization (WHO) reported that only in Asia, Africa and America over 200 million Giardia lamblia infections are reported yearly (Macotela et al. 2004). Contamination with this pathogenic protozoan leads to malnutrition, diarrhea, nausea, cramps, jaundice, avitaminosis, and weight loss in children (Gadelha et al. 2005). The drug of choice in the treatment of giardiasis is metronidazole (Flagyl), which is among the nitroimidazole derivatives and effective on anaerobic bacteria and protozoa. Other drugs used to treat giardiasis are furazolidone and quinacrine (Harris et al. 2001a; Calzada and Alanis 2007). Metronidazole has been used for several decades in the treatment of protozoan, bacterial and fungal infections; nonetheless, there have been some reports regarding the carcinogenic and teratogenic effects on laboratory rats and drug resistance to some isolates of Giardia. Unpleasant side effects including headache, nausea, dizziness, and metallic taste in the mouth have been reported in patients treated with metronidazole (Harris et al. 2000, 2001a). In order to invent appropriate alternative drugs with low side effects and high efficacy, studies on various plants have been started.

Satureja khuzestanica, an indigenous plant widely found in southern parts of Iran, is known for its antiseptic and painkilling health benefits in traditional medicine. In recent years, its antiviral, antibacterial, antifungal, anti-inflammatory, antispasmodic, anti-diarrheal, and vascular dilatation effects of various species of this plant have been reported in different regions of the world (Haeri et al. 2006). Chemical analyses carried out on S. khuzestanica have shown that its effective substance is Carvacrol that constitutes most of the compound (Shahsavari et al. 2009). Garlic (Allium sativum) has been used by humans for many years due to its numerous health benefits. According to old documents, central Asia and China are said to be the origin of this plant due to its properties. The plant has a wide range of medicinal properties. It not only is antibacterial, antiviral, antifungal and anti-protozoan, but also it has beneficial effects on the cardiovascular and human immune systems (Harris et al. 2000, 2001b). Most of these properties of garlic are attributed to Allicin, its effective substance (Harris et al. 2000; Ankri and Mirelman 1999). In addition to its fatality effect on various pathogens, the extract prepared from olive tree leaves can reduce blood pressure and cholesterol as well. Olive leaf extract properties are due to the effectiveness of its component called Oleuropein. The extract of the leaf of this plant has anti-bacterial, anti-viral, anti-fungal and anti-protozoan effects. Some common pathogenic parasites destroyed by the plant in vitro include amoeba, Cryptosporidium Spp., Giardia Spp., Enterobius vermicularis, tapeworms, nematodes and spiral worms (Paliva-Martines 2001).

Considering the disadvantages of metronidazole as the first-line treatment of giardiasis and in order to access natural medicines with limited side effects and high efficacy, the effects of the hydroalcoholic extracts of S. khuzestanica, A. sativum, and olive leaf were evaluated on G. lamblia cysts in vitro and their effects were compared with the effect of metronidazole.

Materials and methods

Preparation of plant extracts and concentrations needed

Fresh leaves of olive and S. khuzestanica collected from Khersdar region in Poldokhtar (south of Lorestan Province, Iran) were washed with distilled water and dried in the shade. The dried leaves were powered using a mill, and the extracts were taken using a Soxhlet extractor and 80° ethanol for olive leaf and 70° ethanol for S. khuzestanica. After concentrating the extracts obtained with a rotary device (vacuum distillation), they were kept and flattened in glass containers and dried at room temperature. Then, the extracts were powdered and kept in completely closed dark glass containers at 4 °C until they were used. The operational efficiency for olive leaf and S. khuzestanica extracts were 25 and 17.5 % respectively. The commercial hydroalcoholic extract of A. sativum (EBN-E-MASOUYEH, Iran) was bought and used.

Evaluation of Giardia cysts sensitivity to the plant extracts

Giardia cysts suspension used in this study was obtained from samples of patients with giardiasis which had been concentrated using the sedimentation technique with normal saline and centrifuged 5 min at 500×g several times. A hemocytometer 5 × 1,000 cyst/ml was applied to determine the number of the cysts. 2 and 5 mg of each extract were added to tubes containing 1 ml of Giardia cysts suspension. Thus, two concentrations (2 and 5 mg/ml) were prepared for each plant extract and were evaluated along with two similar concentrations of metronidazole at two temperatures of 4 and 37 °C and at the 2nd and 4th h after exposure. The bioavailability of G. lamblia cysts were examined using 0.1 % eosin vital staining and a hemocytometer with 40× magnification of an optical microscope. Eosin stain solution accompanied by Giardia cysts suspension, and Giardia cysts suspension alone were considered as the control groups of the study. All the examinations were repeated three times and the percentage of the dead cysts was calculated via the following formula:

$$\left( {{\text{D/}}\left( {\text{L + D}} \right)} \right) \, \times { 100}$$

L is the number of living cysts, D is the number of dead cysts.

Statistical analysis of the data

After completing the tests, the data were analyzed using the statistical test of multiway analysis of variance (ANOVA), Tukey’s test and the SPSS software, version 18. Incidentally, all the tests were performed considering the significance level of 0.05.

Results

The results of multiway analysis of variance statistical test indicated that there was a significant relationship between the variables of type of plant extract/drug (P < 0.001), concentration of plant extract/drug (P = 0.002), exposure time (P = 0.012), and temperature (P = 0.011), on the one hand, and the percentage of dead cysts, on the other hand. The examinations showed that that olive leaf extract had the greatest fatality rate on Giardia cysts in vitro (37.90 %), followed by S. khuzestanica (32.52 %). 250 mg metronidazole pills had relatively effective effect on Giardia cysts in vitro (28.75 ± 10.30 %), while the lowest effect was observed for A. sativum with a fatality rate of 22.65 ± 10.47 %. However, Tukey’s multiple comparison test revealed that the fatality rate of olive leaf extract was clearly and solely more than the effects of the other extracts/drug. The extracts of S. khuzestanica and metronidazole fell in a similar group in terms of the percentage of fatality rate on cysts, and A. sativum extract fell in a separate group alone. Moreover, the results of multiway ANOVA showed an interaction between type of extract/drug and concentration of extract/drug (P = 0.007), so that in all the extracts/drugs, except olive leaf extract, the fatality rates increased with an increase in concentration. No significant interactions were found between the other factors of the experiment. The details of the multiway ANOVA test are presented in Table 1.

Table 1 Factors affecting the percentage of fatality rates on cysts based on multiway ANOVA test
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Olive leaf extract with a concentration of 2 mg/ml, had the most fatality rate on Giardia cysts in vitro 4 h after exposure at a temperature of 37 °C (45.34 ± 0.42 %). S. khuzestanica with a concentration of 5 mg/ml, had an effective fatality rate on Giardia cysts in vitro 4 h after exposure at 4 °C (38.05 ± 5.66 %). The highest fatality rate on Giardia cysts for A. sativum was found with a concentration of 5 mg/ml in vitro 4 h after exposure at 37 °C (36.69 ± 6.99 %). Also, the highest fatality rate for 250 mg metronidazole pills in vitro was found with a concentration of 5 mg/ml 4 h after exposure at 4 °C (34.86 ± 4.57 %). The details of the experimental groups in terms of concentration, exposure time and temperature are presented in Figs. 1, 2, 3 and 4.

Fig. 1
figure 1

Percentage of dead cysts in terms of type of extract/drug and concentration

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Fig. 2
figure 2

Percentage of dead cysts in terms of type of extract/drug, temperature, and concentration

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Fig. 3
figure 3

Percentage of dead cysts in terms of type of extract/drug, exposure duration, and concentration

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Fig. 4
figure 4

Percentage of dead cysts in terms of type of extract/drug, exposure duration, and temperature

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Discussion

Many studies have been conducted to evaluate the anti-parasitic properties of various plants as follows:

In 2008, Shahabi et al. evaluated the effect of the extract and essence of Zenian on Giardia cysts and showed that the minimum inhibitory concentrations (MIC) of the hydroalcoholic extract and essence of Zenian were 100 and 8 mg/ml respectively (Shahabi et al. 2009). The results of a study by Sadjadi et al. in 2006 revealed that the vinegar from grapes had fatality rate on Giardia cysts (Sadjadi et al. 2006). Saffarharandi and his colleagues in a study conducted in 2006 showed that the chloroform extract of garlic on Giardia cysts was completely effective (Saffarharandi et al. 2006). Nazari et al.’s study showed that the hydroalcoholic extract of S. khuzestanica could dramatically reduce glucose and triglycerines levels in rats (Nazari et al. 2005). The results of Soffar et al.’s study in 1991 revealed that garlic extract in children with Giardia and Hymenolepiasis nana was effective and had short treatment periods (Soffar and Mokhtar 1991). Lun et al. attributed the anti-parasitic effect of A. sativum extract with a 14 ug/ml IC50 to diallyl trisulphate, the final stable product resulting from deformation of Allicin (Lun et al. 1994). In Malek et al.’s study carried out in 2006, similar results were reported for metronidazole and furazolidone in the treatment of giardiasis in children (Malek et al. 2006). In our study, after the tests were completed, it was found that olive leaf had the highest effect on Giardia cysts in vitro, followed by S. khuzestanica and metronidazole. In this study, unlike other previous studies, A. sativum extract had relatively a weak effect on G. lamblia cysts (Azadbakht et al. 2003; Behnia et al. 2008). This weak effect can be attributed to the commercial preparation of the extract from drug companies, different methods used by the companies for preparing and processing the extract, the effect of environmental conditions on A. sativum extract compound until consuming time, and diverse species of A. sativum in other studies. In Azadbakht et al.’s study, the IC50 of the extract A. sativum on Giardia cysts was 0.2 mg/ml (Azadbakht et al. 2003). Behnia et al. showed in their study that the original extract of A. sativum had a high anti-amebic effect in a low minimum inhibitory concentration (MIC) (Behnia et al. 2008). Increasing concentrations of plant extract/drug can not generally increase the fatality rate on cysts unless the type of extract/drug is taken into consideration. For example, olive leaf extract with a concentration of 2 mg/ml had the greatest effect on the fatality rate on Giardia cysts. Olive leaf extract probably has a fatality threshold on Giardia cysts, so that if the concentration of the extract is more than the threshold, the fatality rate on the cysts will not increase any more. In other words, the fatality rate of olive leaf extract on Giardia cysts can be increased only to certain concentrations. Increasing exposure duration increases the fatality rates on the cysts, regardless of the type of extract/drug. Generally, except for S. khuzestanica, an increase in temperature increases the fatality rates on the cysts because, contrary to our expectations, in S. khuzestanica extract the fatality rate decreased with increasing temperature at the exposure time of 2 h. It can be due to the negative effect of temperature on the effective components of S. khuzestanica extract. Since S. khuzestanica is a plant native to Iran found in specific regions, very few studies has been conducted on the anti-parasitic properties of this plant. However, other researches have confirmed the fatality property of other species of this plant on culicidae mosquito larvae (Michaelakis et al. 2007). Generally, in eosin solution and Giardia cysts suspension it can not be claimed that the increasing rate of temperature increases the fatality rate on cysts. Contrary to our expectations, at exposure duration of 4 h the fatality rates decreased with an increase in temperature, and the reason is unknown. Some recommendations for subsequent studies are as follows:

  1. 1.

    Lower concentrations of olive leaf extract on Giardia cysts can be compared.

  2. 2.

    The differences in some of the variables of the study including the effect of concentration of extract/drug, temperature, and exposure time on fatality rate can be reevaluated.

  3. 3.

    The effect of the extracts of olive leaf, S. khuzestanica, and A. sativum can be studied on G. lamblia in vivo.