• Anti leishmania effect of pistacia Atlantica subspecies Kurdica on leishmania major
  • Sogol Atefi,1,* Ghazale Adhami,2
    1. Veterinary Graduate.Factulty of Veterinary Medicine,Sanandaj Branch,Islamic Azad University,Sanandaj,Iran.
    2. Assistant Professor.Department of Veterinary Pathobiology,Snandaj Branch,Islamic Azad University,Sanandaj,Iran.


  • Introduction: Leishmaniases are a group of diseases caused by several species of flagellated protozoan parasite, Leishmania, being primarily transmitted through the bite of female phlebotomine sand flies. Approximately 350 million individuals inhabiting in 88 countries in the tropics and subtropics are affected by leishmaniases.(7) A number of risk factors may be involved in the increase of exposure to the sand flies and subsequent Leishmania-associated morbidity, including expansion of urban areas along with forest destruction and migration of people to the endemic regions. Based on the involved sites of the body, clinical disease manifests as cutaneous leishmaniasis (CL), mucocutaneous leishmaniasis (MCL) and visceral leishmaniasis (VL). Based on the published literature, CL is the most common form, with the annual incidence rate of 0.7 – 1.2 million cases. Most of the CL cases have been reported in six countries, comprising Brazil, Columbia, Algeria, Afghanistan, Iran and Syria.(6) In Iran, CL is a seemingly prevalent parasitic infection, with almost 20,000 new cases annually, that are being sorted into two major forms: i) anthroponotic cutaneous leishmaniasis (ACL) due to Leishmania tropica (L. tropica), transmitted by Phlebotomus sergenti (Ph. sergenti) and resulting in dry sores, ii) zoonotic cutaneous leishmaniasis (ZCL) caused by L. major, transmitted by Ph. papatasi and yielding secretory, wet sores. Altogether, successful treatment of CL requires long-lasting administration of obsolete, toxic drugs, i.e., pentavalent antimonials such as sodium stibogluconate and meglumine antimoniate, which may be associated with the emergence of drug-resistance isolates. Other new and efficacious therapeutic options such as amphotericin B are expensive and/or demonstrate lower efficacy.(7,1) Such concerns have directed researchers towards research and development (R&D) on the much cheaper and more efficient compounds. Medicinal plants represent a huge repertoire of therapeutic compounds for healing purposes of different types of wounds, including those related to CL. About 250,000 medicinal plant species have been documented around the world, while only 6% have been characterized biologically and functionally. Herbal medicine is much better accepted globally than synthetic medications; thus the discovery of a plant-based therapy for CL lesions is of utmost importance.(6,14) According to the recent findings, the anti-leishmanial activity of quinoline, flavonoids, saponins, terpens, tetralenes and alkaloids derived from medicinal plants have been shown on some Leishmania species. (15,14,10) The present study was done to evaluate the anti-leishmanial efficacy of the extract of a pistachio tree, called Pistacia atlantica, subspecies Kurdica on L. major lesions in mice, in comparison with the standard glucantime treatment.
  • Methods: 2.1. Parasite culture and maintenance Standard L. major strain (MRHO/IR/5/ER) was retrieved from the Parasitology Department of Tarbiat Modares University, Tehran, and cultured in the Roswell Park Memorial Institute (RPMI-1640) medium supplemented with 10% fetal bovine serum (FBS), 100 IU/ml penicillin and 100μg/ml streptomycin. Culture flasks were incubated at 24±1 oC and checked using an invert microscope on a routine basis. 2.2. Induction of Leishmania lesions Upon reaching the stationary phase, the Leishmania promastigotes were enumerated and a 0.2 ml volume of the parasites (containing 106 promastigotes) was prepared and injected into the base of the tail of BALB/c mice. About 25 days later, lesion was formed at the injection site(FIGURE1) . In order to confirm the presence of Leishmania within lesions, direct sampling was done on glass slides, fixed with methanol, air-dried and stained using Giemsa solution and subsequently examined using light microscopy (100× magnification). 2.3. Treatment groups For this aim, 30 BALB/c mice were sorted into the following groups: 5% plant-based ointment (5 mice), 25% plant-based ointment (5 mice), 50% plant-based ointment (5 mice), glucantime therapy (5 mice), negative control (5 mice) and eucerin (5 mice). A control group was considered for the evaluation of lesion progression, length, recovery, animal mortality and the presence of parasites. All mice in experimental groups were checked regarding disease progress or recovery, length and duration of lesions, presence of the parasites as well as mortality. The diameter of each lesion (millimeter) was measured at first and in the end of the experiment using a caliper. The parasite burden in the lesions was determined during 6 weeks using a grading system for Leishmania lesions, initially (before treatment) and at the end of the experiment (before killing mice). For this purpose, the lesions were sanitized using 70% ethanol and samples were prepared from the exudates using a lancet. The preparations were air-dried, fixed with methanol, stained using Giemsa solution and then assessed regarding the parasite burden, as follows: no parasite per 10 cultures (negative), one parasite per 10 cultures (1+), 1-10 parasites per 10 cultures (2+), 10-100 parasites per 10 cultures (3+), 100-1000 parasites per 10 cultures (4+), and over 1000 parasites per 10 cultures (5+ and more). 2.4. Statistical analysis The significance level of the obtained results among experimental groups was evaluated using One-way ANOVA test and SPSS v21.0 software (SPSS Inc., Chicago, IL, USA). The difference between experimental and control group was assessed using T-test. Of note, a P<0.05 was considered as statistically significant. (3)
  • Results: 3. Results This experiment was conducted during 6 weeks and the mean ± standard deviation (SD) of lesion measurement at the end of each week is provided in Table 1. Based on one-way ANOVA statistics, it could be inferred that there was a significant difference regarding time and treatment in the wound diameter. With respect to the calculated P-value for time variable (P < 0.05), there was statistically remarkable difference among different time periods, so that the mean of the measured lesion was different regarding 6 weeks of experiment, and there observed a statistically significant difference in terms of the time of lesion measurement. Based on Figure 2, there was a statistically significant difference in the diameter of the CL lesions during 6 experimental weeks, while in eucerin-treated group the diameter of lesions even increased during this period. It was deduced that in all treatment groups there was a statistically significant difference between sampling time and the parasite load in lesions. In all groups except of eucerin the parasite load was the highest in the first week and the least in the sixth week. The results corroborated that the parasite load differed in the treatment groups based on sampling time in each week. It was, also, noteworthy that the highest and lowest parasite load belonged to eucerin and 50% Pistacia atlantica extract ointment groups, respectively.
  • Conclusion: The results of the present study along with previous findings shows that Pistacia atlantica plant extract possesses potent leishmanicidal activities in vitro and in vivo. Also, it is recommended to determine the efficacy of different concentrations of Pistacia atlantica extract along with various routes of administration in the human CL cases.(3) Additionally, the prediction, isolation, purification and application of the effective compounds found in the Pistacia atlantica extract is highly suggested for future studies.
  • Keywords: Leishmania major Promastigotes Pistacia atlantica