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Table of Contents
RESEARCH ARTICLE
Year : 2018  |  Volume : 55  |  Issue : 2  |  Page : 111-115

Potent antileishmanial activity of chitosan against Iranian strain of Leishmania major (MRHO/IR/75/ER): In vitro and in vivo assay


1 Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran
2 Molecular and Cell Biology Research Center, Department of Parasitology and Mycology, School of Medicine, Mazandaran University of Medical Sciences, Sari, Iran

Date of Submission29-May-2017
Date of Acceptance18-Apr-2018
Date of Web Publication1-Oct-2018

Correspondence Address:
Mehdi Mohebali
Department of Medical Parasitology and Mycology, School of Public Health, Tehran University of Medical Sciences, Tehran
Iran
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0972-9062.242557

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  Abstract 

Background & objectives: Leishmaniasis is one of the major neglected zoonotic parasitic diseases whose treatment and control is very complex. Pentavalent antimonials remain the primary drugs against different forms of leishmaniasis, however, resistance to antimony and its toxic effects has necessitated the development of alternative medications such as use of medicinal plants and natural compounds. The aim of the current study was to assess the in vitro and in vivo activities of chitosan as a natural resource against Leishmania major.
Methods: Low molecular weight chitosan, with 95% degree of deacetylation was melted in normal saline to a final concentration of 50, 100, 200 and 400 μg/ml. Then, the promastigotes of L. major (Iranian strain) were added to the wells of 96-well plate and 20 μl of each concentration was added to the RPMI 1640 medium. Live and dead promastigotes were counted after adding 0.1% eosin stain. The efficacy of the chitosan was also examined in BALB/c mice infected with Iranian strain of L. major. All in vitro experiments were performed in triplicate and the results of in vitro and in vivo tests were compared to the acetic acid and NaOH as negative control and glucantime as positive control.
Results: The low molecular weight chitosan was completely effective at concentrations of 100, 200 and 400 μg/ml on promastigotes of L. major after 180 min of application. Moreover, in the in vivo study, the mean size of dermal lesions significantly decreased in the groups treated with the chitosan compared to the control group.
Interpretation & conclusion: According to the results of the study, it can be concluded that chitosan is a potent active compound against L. major and could be evaluated as a new antileishmanial drug in the future.

Keywords: Chitosan; in vitro; in vivo; Iran; Leishmania major


How to cite this article:
Esboei BR, Mohebali M, Mousavi P, Fakhar M, Akhoundi B. Potent antileishmanial activity of chitosan against Iranian strain of Leishmania major (MRHO/IR/75/ER): In vitro and in vivo assay. J Vector Borne Dis 2018;55:111-5

How to cite this URL:
Esboei BR, Mohebali M, Mousavi P, Fakhar M, Akhoundi B. Potent antileishmanial activity of chitosan against Iranian strain of Leishmania major (MRHO/IR/75/ER): In vitro and in vivo assay. J Vector Borne Dis [serial online] 2018 [cited 2018 Oct 21];55:111-5. Available from: http://www.jvbd.org/text.asp?2018/55/2/111/242557




  Introduction Top


Leishmaniasis is a vector-borne zoonotic disease, caused by an intracellular protozoan belonging to the genus Leishmania[1],[2]. In spite of several control efforts, the disease is still an important public health problem, especially in the developing countries[1],[3]. An estimated 1,500,000 new cases of cutaneous (CL) and ~500,000 visceral leishmaniasis (VL) cases are reported annually. About 20,000–40,000 deaths/year are associated with leishmaniasis worldwide and the population at risk is around 350 million people[4],[5],[6]. Iran is one of the important foci of CL and L. major is the main causative agent. Out of 31 provinces in the Islamic Republic of Iran, 17 provinces are reported to be affected by this disease[7]. Recently, the incidence of leishmaniasis is reported to be on the upswing because of uninterrupted increase in the number of vectors of the disease mainly due to global warming[7],[8]. Pentavalent antimonials, of the several medications, are considered as the first line drugs in the treatment of CL and VL in majority of the countries, but due to toxicity, development of resistance in vectors, and high cost; its use is limited in undeveloped and developing countries[1],[10]. Thus, new remedies for the treatment of leishmaniasis are instantly needed[11],[12].

Although, over 25 new substances are described as acting against Leishmania spp, only few of them are reported to be effective for use in humans[13]. Most of them are used in parenteral form, but there are a lot of side effects when using oral for[14],[15]. Several species of insects and their products are considered for its treatment in the traditional medicines which are potentially useful for modern medicine; however, there is limited information about them in the scientific literature/reports[16].

Chitin, a type of carbohydrate of animal animal origin is found abundantly in nature and forms the basis of the outer skeleton of insects and crustaceans like shrimp, crabs, and lobsters[17]. Chitosan is derived from chitin, which is characterized by its widespread applications in the form of antibacterial, antiviral, antifungal and antiparasitic agents[18]. It has been reported to have broad spectrum of activity and high killing rate against wide range of organisms including algae, bacteria, yeasts, fungi, and parasites in the in vivo and in vitro experiments, while there is not any report about its toxicity for the mammalian cells[19],[20]. Dieseline chitosan hydrogel formulation was used to treat L. major-infected BALB/c mice and the chitosan formulation was incapable to slow the lesion progression and reduce the parasite burden[21]. Considering the inadequacy of currently available treatments, their side effects and the fact that some species of Leishmania are resistant to the existing drugs, exploration of new medications are necessary in order to find a more selective and effective treatment with fewer side effects. Thus, this study aimed to evaluate both in vitro and in vivo antileishmanial effects of chitosan.


  Material & Methods Top


Chitosan solution preparation

Chitosan with low molecular weight and viscosity of 200–800 cP, (Sigma-Aldrich, St. Louis, MO, USA) and with 95% deacetylation degree was liquefied in normal saline with 1% (w/v) acetic acid to achieve the concentration of 50, 100, 200 and 400 μg/ml[28]. All concentrations were kept overnight at room temperature to reach complete dispersion. The pH of all tubes was then adjusted to seven by adding 1 mol NaOH[23].

Parasites

The promastigotes of L. major (Iranian strain MRHO/IR/75/ER) was obtained from the Department of Parasitology and Mycology, Tehran University of Medical Sciences, Tehran, Iran. The promastigotes were transferred into RPMI 1640 medium supplemented with 20% fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, MO, USA), 25 mM of HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid], 100 U/ml of penicillin, and 100 μg/ml streptomycin (Sigma-Aldrich, St. Louis, MO, USA), and incubated at 24 °C for 72 h[24].

In vitro assay

Promastigotes at log phase were centrifuged at 2000 rpm for 5 min, diluted in fresh culture medium to a final density of 2 × 106 cells/ml. From the parasite suspension, 200 μl were added to the different wells, and then 20 μl of each concentration of the chitosan was added to these wells. Two rows of 96-well plate left blank for 1% (w/v) acetic acid and 1 mol NaOH as the negative controls. Live and dead promastigotes were counted after adding 0.1% eosin stain with a light microscope[25].

In vivo assay

In total 15 female BALB/c mice (6–7 wk old) purchased from Razi Rad Industries were used in the current investigation. All mice were inoculated subcutaneously in the base of the tail with approximately 1.6 × 106 promastigotes of L. major in the stationary stage. Mice were divided into three five-membered groups based on the activity obtained in in vitro assay. The groups were categorized as follows; group 1 was treated with 200 μg/ml of commercial chitosan; group 2 was treated with 400 μg/ml of commercial chitosan; group 3 was left untreated (control group). Treatment was started when local lesions were obvious. Topical treatment was applied on the mice on every 28 continuous days. The lesion size was measured weekly before and after treatment by dial micrometer (Starrett Dial indicator, model 25A, USA) in two diam (a and b) and comparing it with that of untreated lesions. The wound size was estimated/calculated by the following formula[26]:



Correspondingly, pre-treatment and 7, 14, 21 and 28 days post-treatment, slides of infected lesions were prepared and fixed with methanol, stained with Giemsa stain and examined by a light microscope (1000×). Chitosan effectiveness was evaluated by comparing the diameters of wounds and the number of slides with amastigotes, between treated and untreated groups.

Ethical statement

The ethical approval for the study was obtained from the Tehran University of Medical Sciences, Iran with Reference No. 1393/25642, dated March 9, 2015.

Statistical analysis

All in vitro experiments were performed in triplicate. The mean and standard error of all experiments were determined. Statistical analysis of the differences between mean values of the experimental groups was done by chisquare, analysis of variance (ANOVA), and paired t-test. The p-value < 0.05 was considered significant.


  Results Top


In vitro assessment

The effect of low molecular weight chitosan on pro-mastigotes of L. major at different concentrations (50, 100, 200 and 400 μg/ml) after 30, 60, 120 and 180 min is presented in [Figure 1] and [Table 1]. In all concentrations, the effect of chitosan was time-dependent, i.e. parasite viability decreased with the passing time. Moreover, the effect of chitosan on L. major was significantly different in the treated mice compared to the negative controls (p < 0.001). In all four time-groups, the growth of L. major decreased with the increasing concentration.
Figure 1: In vitro effectiveness of chitosan at concentrations of 50, 100, 200 and 400 μg/ml on promastigotes of Leishmania major after 30, 60, 120 and 180 min.

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Table 1: The effects of chitosan (in vitro) at different concentrations on promastigotes of L. major after 30, 60, 120 and 180 min, in comparison to 1% acetic acid and 1 mol NaOH as negative controls and glucantime as positive control

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In vivo assessment

The mean size of lesions decreased in the groups treated with commercial chitosan in comparison with the control group. The mean lesion size in the control group after 28 days of intervention was 8.47 mm, whereas it was 2.07 and 1.05 mm in groups treated with the 200 and 400 μg/ml of chitosan, respectively. The mean difference between treated and untreated controls was significant (p < 0.05), while there was no significant difference between the efficacy of the 200 and 400 μg/ml (p > 0.05), as evident from [Table 2].
Table 2: The effects of chitosan at concentration of 200 and 400 μg/ml in BALB/c mice infected with Iranian strain of L. major after 7, 14, 21 and 28 days of treatment (in vivo), in comparison to controls groups.

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  Discussion Top


The antimicrobial activity (antibacterial, antiviral, and antifungal) of low molecular weight chitosan has been described in many reports, but limited efforts have been made to define the antiparasitic potential of chitosan. Chitosan is biodegradable, safe, and has a wide variety of applications against several pathogens[18],[19],[20],[21]. The mentioned properties, along with the non-toxicity profile, make chitosan favourable for the pharmaceutical products for present and future uses[22], [27],[28].

Eaton et al[29] have reported that >0.025% of chitosan inhibits the growth of fungi and bacteria including Helminthosporium, Fusarium, Alternaria and Escherichia coli. A comparable study exhibited that 1.250 μg/ml chitosan after 360 min of exposure could completely stop the viability of Trichomonas gallinae[30],[31]. Contrary to this study, a novel dieseline chitosan hydrogel formulation used in the treatment of L. major infected BALB/c mice by Schwartzthe et al[21] indicated that diselenide chitosan was ineffective to slow down the lesion progression or decrease the parasite burden. There are insufficient reports about antiparasitic effects of chitosan and hereby in this study, we report chitosan as an anti-L. major agent. The results showed a high promastigocidal effect of chitosan in comparison with control groups at the concentrations of 50, 100, 200, 400 μg/ml after different exposure times (30, 60, 120 and 180 min). The maximum mortality rate (100%) was achieved at 100, 200 and 400 μg/ml concentrations after 180 min of exposure time.

The in vivo results of this study indicated that low molecular weight chitosan has significant effect on wound healing, though the wound in some cases did not heal completely. There was a significant difference in wound healing between case and control groups (p < 0.005).

Rahimi-Esboei et al[32] used the chitosan isolated from Penicillium viridicatum, and P. aurantiogriseum against protoscolices of hydatid cyst and compared with commercial chitosan. They concluded that among the different types of chitosan, commercial chitosan has 100% scolicidal activity in vitro. Similarly, Yarahmadi et al[33] carried out a survey on the effect of chitosan on the viability of Giardia lamblia cysts and reported 100% mortality rate at the concentration of 400 μg/ml after 180 min of exposure.

Pentostam, glucantime, amphotericin B, pentamidine, and paromomycin are the drugs used in first and second-line treatments for CL, but development of drug resistance in some species is the main challenge in the treatment[34],[35],[36],[37]. Additionally, the conventional drugs of choice for leishmaniasis are expensive, toxic, need a long time parenteral administration, and have variable efficacy[25],[27]. Natural compounds are an essential foundation in the search for new therapeutic options.


  Conclusion Top


Based on the results of the present study, it can be concluded that chitosan is a potent active compound against L. major and could be evaluated as a new antileishmanial drug in the future. Chitosan is easily accessible and can be readily prepared in economical manner. Consequently, it may be a promising treatment for leishmaniasis after adequate studies. Nevertheless, in vivo effectiveness of chitosan on humans and the feasible side effects and toxicity needs further investigations.

Conflict of interest

The authors report no conflicts of interest in this work.


  Acknowledgements Top


This study has been financially supported by the Tehran University of Medical Sciences, Tehran, Iran (Grant No. 25642-61-02-93). The authors would like to express their deep thanks to all the staff of the Leishmaniasis Laboratory in Tehran University of Medical Sciences, Tehran, Iran for their help and assistance.



 
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    Tables

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