Anti-Toxoplasma activities of methanolic extract of Sambucus nigra ( Caprifoliaceae ) fruits and leaves

Toxoplasma gondii is an obligatory intracellular parasite that infects a wide range of warm-blooded animals and humans. Considering the severity of toxoplasmosis, side effects of current treatments, and the contribution of the ethnopharmacological knowledge for the treatment of parasitic infections, the aim of the present study was to investigate the efficacy of methanolic extracts from the fruits and leaves of Sambucus nigra against tachyzoite of T. gondii. For this, fruits and leaves of S. nigra were collected from Mazandaran province, Iran, were dried under the shade, and powdered using a commercial electrical blender. For extractions, methanol was used as solvent. virulent RH strain of T. gondii was maintained in mice and macrophages containing tachyzoites were aspirated from the peritoneal cavity. Four concentrations (5, 10, 25 and 50mg/mL) of S. nigra extract were incubated with infected macrophages for 30, 60, 120 and 180 minutes and the viability of the tachyzoites were evaluated by trypan blue staining. Results showed that S. nigra fruit extracts at the concentrations of 5 and 10mg/ mL killed 100% of T. gondii tachyzoites after 60 and 120 minutes, respectively; and concentrations of 25 and 50mg/mL killed 100% of the tachyzoites after 30 minutes. Additionally, extract of S. nigra leaves, at the concentrations of 5, 10 and 25mg/mL after 180 minutes, and concentration of 50mg/mL after 60 minutes, resulted with the highest efficacy. Our results showed that S. nigra has acceptable efficacy in vitro and the parasiticidal effect of fruit extract was significantly better than leaf extract. However, in vivo efficacy of this extract needs further investigation. Rev. Biol. Trop. 63 (1): 7-12. Epub 2015 March 01.

Toxoplasma gondii is an obligatory intracellular protozoan parasite, with wide prevalence in humans and a broad spectrum of vertebrate hosts.It is attained by ingestion of oocysts from contaminated food or water with feces of cats or the ingestion of tissue cysts in under cooked meat (Petersen, 2007).Toxoplasmosis in most situations is an asymptomatic disease, but in some people, flu-like symptoms, swollen lymph glands, muscle aches and pains that may last from a few days to several weeks has been experienced.Toxoplasmosis is an endemic disease which emerged as one of the most common opportunistic infections in immunocompromised patients (Weiss, & Dubey, 2009).Especially in people with HIv infection, toxoplasmosis commonly involves the brain and causes encephalitis.It may also cause death in these patients (Nissapatorn, 2009).During pregnancy, T. gondii can be transmitted through the placenta and may result in congenital toxoplasmosis, and probably, fetal death.Affected infants may suffer a variety of serious clinical manifestations at birth, including nervous system involvement, mental retardation, neonatal growth retardation, ocular defects, blindness in later life and other generalized clinical manifestations (Ambroise-Thomas, & Pelloux, 1993).
The recommended drugs for treatment or prophylaxis of toxoplasmosis are Pyrimethamine and Sulfadiazine; however, the available drugs are not completely effective in killing the parasite (Serranti, Buonsenso, & valentini, 2011).On the other hand, these drugs have side effects such as neutropenia, severe drop of platelet count, thrombocytopenia, leucopenia, elevation in serum creatinine and serum liver enzymes, haematological abnormalities and hypersensitivity reactions (Bosch-Driessen et al., 2002;Silveira et al., 2002;Schmidt et al., 2006).For many centuries, herbal remedies and plant extracts have been used as treatments for ailments from headaches to parasite infections (Jones, 1996).Only in the past 20-30years, scientists have seriously begun to determine the efficacy of plant-derived traditional remedies and their mode of action.Sambucus spp.has been reported to be an insect repellent, antihemorrhoid, anti-protozoa, and anti-bacterial treatment.It is also mentioned for treatment of burns and infected wounds, edema, eczema, common cold, inflammation and rheumatism (Ahmadiani, Fereidoni, Semnanian, Kamalinejad, & Saremi, 1998;Yeşilada, Gürbüz, & Shibata, 1999;Rahimi-Esboei, Ebrahimzadeh, Gholami, & Falah-Omrani, 2013).Considering the side effects of anti-Toxoplasma drugs and the severity of toxoplasmosis, particularly in the fetus and the contribution of the ethnopharmacological knowledge for the treatment of parasitic infections, it is necessary to investigate on new anti-Toxoplasma compounds with high activity, low toxicity, and that are cheaper and more effective.Therefore, the aim of the present study was to survey on the efficacy of methanolic extracts from the fruits and leaves of S. nigra on tachyzoites of T. gondii.

Preparation of S. nigra extract:
The fully ripped fruits and leaves of S. nigra (from seven plants of 6 years old; height: 3-4.5m) were collected in October 2012 from Mazandaran province, Iran.This province is in the Caspian Sea littoral and is located in the North part of the country that has moist climate.The plants were dried under shade, and powdered mechanically using a commercial electrical blender (Pars Khazar BG-310P).To obtain the methanolic extract, 150g of fruit and leaves dried powders were added to 350mL of pure methanol (30-35 o C) and mixed gradually for 1h using a magnetic stirrer.The obtained solutions were covered and left at room temperature (22-25 o C) for 24h.The solutions were stirred again and filtered using Whatman No.1 filter paper and the resulting extracts were concentrated over a rotary vacuum (Heidolph-Laborota 4000 efficient, Germany) at 35°C, until a crude solid extract was obtained.The remaining semisolid material was then freeze-dried at -50ºC for 24h (MPS-55 Freeze-drier, Cperon, Korea).The obtained filtrate (14.5g) was placed into a sterile glass container and stored at 4 o C for further use (Rahimi-Esboei et al., 2013).

Effect of S. nigra extracts on tachyzoites:
In this study, four concentrations (5, 10, 25 and 50mg/mL) of the methanolic extracts from the fruits and leaves of S. nigra were assessed for 30, 60, 120 and 180minutes.Two mL of each concentration and 4×10 6 fresh tachyzoites were placed in test tube.The contents of the tubes were gently mixed, then incubated at 37 o C for 30, 60, 120 and 180minutes.In the end of each incubation time, two mL of 0.5% trypan blue stain was then added to the settled tachyzoites.Then was smeared on a glass slide, and examined under a light microscope.The percentage of dead tachyzoites was determined by counting 200 tachyzoites.Tubes containing PBS and pyrimethamine in concentration of 100mg/mL were considered as negative and positive control groups, respectively.The experiments were performed in triplicate.
Statistical analysis was performed using SPSS-ver20 software.Differences between test and control groups were analyzed by repeated measures ANOvA test.A p value less than 0.05 were considered statistically significant.

RESULTS
The anti-Toxoplasma effects of the extract of S. nigra's fruits are summarized in Table 1.This extract at the concentration of 5mg/mL killed 62.71, 90.58 and 100% of tachyzoites after 30, 60 and 120 minutes, respectively.Concentration of 10mg/mL after 60 minutes, and also concentrations 25 and 50mg/mL after 30 minutes killed 100% of the tachyzoites.
The anti-Toxoplasma effect of methanolic extracts from the fruits of S. nigra was extremely significant compared to the negative control group in all exposure times (p<0.05),and a significant difference was found between the fruit methanolic extract of S. nigra and the positive control after 30 and 60minutes in all concentrations.The anti-Toxoplasma effects of the extract from S. nigra's leaf were summarized in Table 2. Leaves extract at the concentrations of 5, 10 and 25mg/mL killed more than 90% of the tachyzoites in 180minutes, while at the concentration of 50mg/mL, 94% of tachyzoites were killed in 60minutes.
The anti-Toxoplasma effect of the methanolic extract from the leaf of S. nigra was extremely significant compared to the negative control group in all exposure times (p<0.05),but they did not show any significant difference compared with positive control.

DISCUSSION
Pyrimethamine, sulfadiazine, and leucovorin are recommended by World Health Organization (WHO, 1995) and the Centers for Disease Control and Prevention (CDC) as the standard care for toxoplasmosis (Petersen, 2007;Rodriguez & Szajnman 2012).These drugs act through inhibiting folic acid synthesis in T. gondii, but as in other chemical drugs these have unpleasant side-effects.Therapeutic plants are the traditional treatment of diseases and they are suggested by WHO (World Health, 1995).The use of therapeutic plants by people in developing countries is common because these products are safe and available at low cost.
Our results showed that S. nigra has acceptable efficacy on tachyzoites of T. gondii in vitro and the tachizoitocidal effects of fruit were significantly better than leaf.According to our results, there was a significantly difference between S. nigra extracts and control groups in all exposure times.Sambucus spp. is a member of the Caprifoliaceae family that contains several ingredients responsible for pharmacological activity.Four species of the genus Sambucus are growing in Iran.Of these species, S. ebulus and S. nigra are extensively seen in the Northern regions.
Artemisia is the most common anti-Toxoplasma extract in the world.De Oliveria et al., (2009) indicated that treatment of T. gondii tachyzoites with A. annua infusion before infection in HFF (Human Foreskin Fibroblast) cells exhibited a dose-response inhibitory curve that reached up to 75% of inhibition.Yang et al., (2011) used aqueous extracts of Astragalus membranaceus and Scutellaria baicalensis on tachyzoites of T. gondii in vitro; they concluded that parasites treated with either A. membranaceus or S. baicalensis had significantly decreased intracellular replication at 72, 96 and 120 hours after treatment.Gozalbes et al., (2000) showed that among the 24 quinolones tested, 20 had positive activity against T. gondii with the IC50s ranged from 0.4mg/mL for trovafloxacin to 100mg/mL for cinoxacin and levofloxacin.
The results of our study showed that methanolic extract of S. nigra fruits is an effective anti-Toxoplasma agent and it seems to be used as a candidate extract for treatment of toxoplasmosis in future researches.However, the in vivo efficacy of this extract needs to be explored.Even though S. nigra is edible, it is possible side effects when used as an anti-Toxoplasma agent needs more investigation.This is the first report on the anti-Toxoplasma activity of S. nigra.

ACKNOWLEDGMENTS
This study received financial support (grant no.91-68) from Deputy of Research, Mazandaran University of Medical Sciences, Sari, Iran.

TABLE 1
The effect of fruits of S. nigra on mortality of T. gondii tachyzoites in vitro Pos. = Positive control group receiving 100mg/mL pyrimethamine.Neg.= Negative control group receiving PBS.