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Antileishmanial activity of essential oil from Chenopodium ambrosioides and its main components against experimental cutaneous leishmaniasis in BALB/c mice.

Introduction

Leishmaniasis are tropical anthropo-zoonotic diseases caused by protozoan parasites of the genus Leishmania. More than 12 million people are affected in 98 countries (Alvar et al. 2012). The clinical manifestations may range from single cutaneous lesions to the fatal visceral leishmaniasis. Currently, there is no vaccine commercially available for use in humans and recommended chemotherapy relies mostly on pentavalent antimonials or amphotericin B, pentamidine and miltefosine as alternatives. However, conventional drugs present several limitations, including toxicity, cost and resistance (Murray et al. 2005).

The genus Chenopodium (Chenopodiaceae) originates from South America and includes 150 species which are widely distributed in hot sub-tropical and tropical, but only few of them have been scientifically investigated. One example is Chenopodium ambrosioides, which have been used for centuries by native people as anti-parasitic (Franca et al. 1996; Quinlan et al. 2002). In previous studies, we reported potential activity of essential oil (EO) from C. ambrosioides against experimental cutaneous leishmaniasis in BALB/c mice caused by Leishmania amazonensis. The aim of this study was to compare the in vivo anti-leishmanial activity of the EO from C. ambrosioides and its major components.

Materials and methods

Parasite strain

The MHOM/77BR/LTB0016 strain of L. amazonensis was kindly provided by the Department of Immunology, Oswaldo Cruz Foundation (FIOCRUZ), Brazil. Parasites were routinely isolated from the mouse lesions and maintained as promastigotes at 26[degrees]C in Schneider's medium (Sigma-Aldrich, St. Louis, MO, USA) containing 10% heat-inactivated fetal bovine serum (Sigma-Aldrich), 100 ([micro]g of streptomycin/ml, and 100U of penicillin/ml.

EO of C. ambrosioides and major components

C. ambrosioides was collected and the EO was extracted as previously described (Monzote et al. 2009). A voucher specimen (No. 4639) was deposited at the Experimental Station of Medicinal Plants "Dr. Juan Tomas Roig", Cuba. The composition of EO was analyzed by high resolution gas chromatography-mass spectrometry (HRGC-MS), using an QP5050-A, Shimadzu DZU, system equipped with flame ionization detection (280 [degrees]C), column 30 m, 0.25 [micro]m film thickness, and 0.25 mm fused silica capillary column SPB-1; a flow rate of 1.5ml/min; a temperature program from 50 [degrees]C, rate 5[degrees]C/min, to 280 [degrees]C for 30 min and with the injection port in split mode 1:40 at 280 [degrees]C; mass spectrometer with ionization energy of 70 eV and ion source at 280[degrees]C.

The Ascaridol (Asc) was obtained by chemical synthesis as previously published (Monzote et al. 2009). Carvacrol (Car) and caryophellyne oxide (Caryo) were obtained from Sigma Aldrich (Vienna, Austria). All products were diluted in dimethyl sulfoxide (DMSO).

Reference drugs

Glucantime (GTM, Rhone-Poulenc Rorer, Mexico) at 30mg/ml was used as reference drug (Fig. 1).

Animals

Female BALB/c mice, with a body weight of approximately 20-22 g, were obtained from The National Center of Laboratory Animals Production (CENPALAB, Cuba) and maintained according to "Guideline on the Care and Use of Laboratory Animals". Protocol to animal use was approval by Ethic Committee from Institute of Tropical Medicine Pedro Kouri (CEI-IPK 13-10), Havana, Cuba.

In vivo experiment

On day 0, normal BALB/c mice received subcutaneous injections in the right hind footpad of 5 x [10.sub.6] stationary-phase L. amazonensis promastigotes. Four weeks post-infection (p.i.) the animals were randomly divided into eight groups of 10 mice each and the treatment was initiated with 25 p,l of EO, Asc, Car or Caryo at a dose of 30mg/kg dissolved in a mixture of 30:70% (v/v) of DMSO:saline solution. In parallel, a group was treated also with 25 [micro]1 of an artificial mix of Asc, Carv and Caryo at a dose of 30 mg/kg of each pure compound. Other two groups were included, one of them treated with GTM at 28 mg/kg dissolved in saline solution: while the othergroup was treated with vehicle (mixture of 30:70% (v/v) of DMSO:saline solution). Treatments were administered by intralesional route daily during 14 days. A group of infected and untreated mice was also included.

Disease progression was monitored weekly by measuring footpads swelling of the lesion diameter between 30 and 60 days pausing a digital caliper. Average lesion size was calculated as the differences obtained between infected and uninfected footpads. On day 45 and 60 p.i., three animals of each group were killed by cervical dislocation and parasite burden determined, using the culture microtitration method (Buffet et al. 1995). Briefly, a sample of the lesion was excised, weighted and homogenized in 4 ml of Schneider's. Under sterile conditions a serial fourfold dilution was prepared in plates with 96-wells. After 7 days of incubation at 26 [degrees]C, plates were examined with an inverted microscope. The final titer was defined as the last dilution for which the well contained at least one parasite. The parasite burden was calculated as follows: parasite burden = (geometric mean of reciprocal titers from each duplicate/weight of homogenized cross section) x 400.

Statistical analysis

Data on lesion progression and parasite burden were analyzed for statistical significance by the analysis of variance test, following of a Post Hoc Test (LDS test or planned comparison) using the STATISTICA for Windows Program (Release 4.5, StatSoft, Inc. 1993).

Results

The HR GC-MS analysis of EO showed a complex mixture. Table 1 shows the time of retention and some characteristic of the principal compound.

The animals treated with Asc, Car and Caryo developed similar lesions (p>0.05) compared with control groups (animals treated with vehicle or untreated mice) until the treatment was finished (Fig. 2A). At the end of experiments (10 weeks p.i.) the mice treated with pure compounds showed statistically significant differences with respect to control groups (p<0.05) and a similar lesion size (p >0.05) as animals treated with GTM. EO prevented lesion development compared (p<0.05) with untreated animals and treated with vehicle. In addition, the efficacy of EO was also statistically superior (p < 0.05) compared with the GTM-treated animals during the last weeks of experiment (8, 9 and 10 weeks p.i.), as well as compared with animals treated with pure compounds. The results were corroborated by measurements of the parasite burden in the footpad by the culture microtitration method on day 45 and 60 p.i. (Fig. 2B). Differences in lesion size between the animals treated with EO, GTM and without treatment could be appreciated in Fig. 2C. Artificial mix of pure compounds caused the death of treated animals after three doses.

Discussion

Highly susceptible BALB/c mice were infected and treated with EO from C. ambrosioides or main pure compounds for determination of their in vivo treatment efficacy. Intralesional route of administration of the products, which is an established technique for anti-leishmanial agents, was selected for these experiments taking into account the high toxicity of the pure compounds previously demonstrated in in vitro assays (Monzote et al. 2009). In parallel, this technique reduces the dosage of product, the risk of systemic toxicity and is appropriate also for outpatient use (Alkawajah and Larbi, 1997).

In the scientific literature, different reports have been demonstrated the useful of intralesional administration in clinical studies, including the GTM (Salmanpour et al. 2006), sodium stibogluconate (El-Sayed and Anwar 2010) and amphotericin B liposomal (Yardley and Croft 1997). In experimental animal models have been also constituting an alternative aplication route in infected mice with Leishmania spp., such the oil from Lippia sidoides (de Medeiros et al. 2011), the natural product licochalcone A (Chen et al. 1994) and a organotellurium compound (RT-01) obtained by chemical synthesis (Cantalupo et al. 2009).

While efficacy for EO was observed, the pure compounds showed a slight transient recrudescence in lesion size between 9 and 10 weeks p.i. In addition to the anti-leishmanial activity of EO and its components, it could be associated with indirect actions, such as antioxidant, antiinflammatory or immunostimulatory activities (Cruz et al. 2007). Patricio et al. reported that the intralesional treatment of infected animals with L amazonensis with an hydroalcoholic extract from C. ambrosioides increased the nitric oxide production and stimulated the hydrogen peroxide in the site of infection, which could be related with a potentiating of microbicide mechanism of macrophage and as consequence increased the efficacy of the product (Patricio et al. 2008).

In our work, the EO showed a better activity when compared to animals treated with GTM, showing statistical differences (p < 0.05) between both groups (weeks 8,9 and 10). GTM or meglumine antimoniate is a synthetic anti-leishmanial drug based on pentavalent antimonial (Sbv) that have been clinically recommended as first line drug to treat the infections by L. mexicana complex in the New World (Murray et al. 2005, Frezard et al. 2009). In our study, L. amazonensis reference strain used is susceptible to GTM and recommended dose was used (Murray et al. 2005). The low effect of glucantime (Nakayama et al. 2005) has been also described previously in animals experimentally infected with L. amazonensis.

In parallel, artificial mix of pure compounds caused the death of treated animals after three doses, which could be cause by an increase of compound toxicity. In our study, same concentration of components in the mix was used with the aim to compare with single agents administered or with EO treatment, which were administered at 30 mg/kg. Further experiments could be addresses in order to: (i) evaluate an artificial mix with the three main constituents in a same ratio of their concentrations in the oil, (ii) perform studies with different concentration of pure compounds with the aim to standardize a dose of compounds safety to mice, (iii) evaluate different administration regimens and (iv) analyzed possible role of different compound combinations in toxicity or anti-leishmanial activity caused. This result also suggests the superiority of EO to use as active principle in an anti-leishmanial formulation compared with pure compounds.

In conclusion, our results demonstrated that the EO from C. ambrosioides showed a better efficacy in comparison with its pure major compounds. Standardization of EO as a natural medication or formulation of an artificial mixture of pure compounds deserves consideration in future studies to develop new therapeutic anti-leishmanial alternative. In addition, the evaluation of EO and pure compounds in combination with synthetic conventional anti-leishmanial drug could contribute to new generation of phyto-pharmaceuticals (Wagner and Ulrich-Merzenich 2009).

ARTICLE INFO

Article history:

Received 10 September 2013

Received in revised form 23 January 2014

Accepted 2 March 2014

Acknowledgments

The award of an Ernst Mach scholarship to Lianet Monzote by the Austrian Exchange Office is gratefully acknowledged.

References

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L. Monzote (3) *, J. Pastor (3), R. Scullb, L. Gille (c)

(a) Parasitology Department, Institute of Tropical Medicine "Pedro Kouri", Havana, Cuba

(b) Department of Chemistry, Institute of Pharmacy and Food, Havana University, Cuba

(c) Biochemical Pharmacology and Toxicology Unit, Department of Biomedical Sciences, University of Veterinary Medicine, Vienna, Austria

Abbreviations: Asc, ascaridole; Car, carvacrol; Caryo, caryophyllene oxide; DMSO, dimethyl sulfoxide; EO, essential oil; GTM, glucantime; HRGC-MS, high resolution gas chromatography-mass spectrometry; p.i., post-infection.

* Corresponding author at: Departamento de Parasitologia, Instituto de Medicina Tropical Pedro Kouri. Apdo No. 601, Marianao 13. La Habana, Cuba.

Tel.: +53 7 255 3637; fax: +53 7 202 0451.

E-mail address: monzote@ipk.sld.cu (L. Monzote).

http://dx.doi.Org/10.1016/j.phymed.2014.03.002
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Title Annotation:Short communication
Author:Monzote, L.; Pastor, J.; Scull, R.; Gille, L.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Date:Jul 15, 2014
Words:2475
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