In vitro efficacy of Coriandrum sativum, Lippia sidoides and Copaifera reticulata against Leishmania chagasi Eficácia in vitro de Coriandrum sativum, Lippia sidoides e Copaifera reticulata sobre Leishmania chagasi

The increased incidence of visceral leishmaniasis (VL) in Brazil is due to a lack of effective disease control measures. In addition to that, no effective treatment exists for canine VL in response to synthetic drugs. Thus, the objective of this study was to evaluate the effect of the essential oils of Coriandrum sativum and Lippia sidoides, and oleoresin from Copaifera reticulata, on Leishmania chagasi promastigotes and amastigotes. We also examined the toxicity of these treatments on the murine monocyte cell line RAW 264.7. To determine the IC50 a MTT test (3-(4,5-Dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide) was performed on promastigotes, and an in situ ELISA assay was conducted on amastigotes. Here, we demonstrate that oleoresin from C. reticulata was effective against both promastigotes (IC50 of 7.88 μg.mL–1) and amastigotes (IC50 of 0.52 μg.mL–1), and neither of the two treatments differed significantly (p > 0.05) from pentamidine (IC50 of 2.149 μg.mL–1) and amphotericin B (IC50 of 9.754 μg.mL–1). Of the three plant oils tested, only oleoresin showed no toxicity toward monocyte, with 78.45% viability after treatment. Inhibition of promastigote and amastigote growth and the lack of cytotoxicity by C. reticulata demonstrate that oleoresin may be a viable option for analyzing the in vivo therapeutic effects of leishmanicidal plants


Introduction
The World Health Organization (WHO) considers visceral leishmaniasis (VL) to be a major tropical zoonotic disease (MISHRA et al., 2009). VL, which continues to resist modern control efforts, is most common in northeastern Asia, eastern Africa and northeastern Brazil, but cases also occur in southern Europe and elsewhere. Each year, there are approximately 500,000 new cases and more than 50,000 deaths worldwide; however, because leishmaniasis is not a commonly reported disease in many countries, these values are probably underestimated (WHO, 2010). In Brazil, this disease has expanded throughout the canine and human populations in various regions (RONDON et al., 2008;BRASIL, 2009). In addition, infected dogs have been associated with all human disease outbreaks, representing the main link in the chain of transmission, and, despite the existence of other Leishmania reservoirs, such as horses and cats, dogs are the only confirmed domestic reservoir (GRAMICCIA; GRADONI, 2005).
Until now, canine visceral leishmaniasis (CVL) has proven to be resistant to available synthetic antiparasitic drugs, thus reinforcing the need for alternatives in disease control. CVL therapies aim to reduce the parasite load, minimize organ damage by the parasites, and redirect the immune response to improve animal health and prevent relapse .
Alternative methods of parasite control, including deltamethrinimpregnated collars or spot-on permethrin-based topical insecticides, are being employed in an attempt to block transmission . Furthermore, the use of plants for new medicines has increased, both to combat multidrug-resistant parasites and to maximize disease control (CROFT; COOMBS, 2003;SHARIEF et al., 2006). Essential oils are natural products of great medical importance because they already possess proven anti-fungi, antimicrobial, and antileishmanial activities (ANTHONY et al., 2005;OLIVEIRA et al., 2009).
The objective of this study was to evaluate the effect of the essentials oils of Coriandrum sativum and Lippia sidoides and oleoresin from Copaifera reticulata on L. chagasi promastigotes and amastigotes, as well as their cytotoxicities in the murine monocyte cell line RAW 264.7.

Extraction of essential oils and oleoresins
The essential oil of L. sidoides was purchased from PRONAT (Produtos Naturais LTDA). C. reticulata oleoresin and C. sativum seeds were purchased at a street fair in the state of Pará and the state of Ceará, Brazil, respectively. C. sativum seeds were dried for one week, and the essential oil was extracted by steam distillation.

Phytochemical tests
Qualitative phytochemical tests of phenols, tannins, catechins, leucoanthocyanidins, flavonoids, steroids, terpenes, alkaloids, and saponins were performed according to Matos (2009) and Siddiqui et al. (2009). These tests are based on the visual observation of colorimetric changes or on the formation of a precipitate after the addition of specific reagents.

Chemical analysis
The oils were chemically analyzed at the Laboratório de Análise de Alimentos, EMBRAPA/Agroindústria using gas chromatography/mass spectrometry (GC/MS) on a 5971 GC/MS (Hewlett-Packard  ) at a temperature of 270 °C. Constituent molecules were identified by their retention indices and mass spectra compared to a database of known chemical signatures (ALENCAR et al., 1984;ADAMS, 1989).
Amastigotes (Laboratório de Protozoologia/USP) were cultured with the murine monocyte cell line RAW 264.7 (Sigma-Aldrich  ) in 96 well microplates. Cells were counted in a Neubauer chamber and plated at a density of 1 ×10 4 cells/well. Promastigotes were added at a ratio 10:1 parasites to cell. Cells were cultured in Dulbecco's medium (Cultilab  ) with 5% FCS, sodium bicarbonate, and 40 mg.mL -1 gentamicin. Bottles were kept ajar and cultivated under glass with 5% CO 2 at 36.6 °C. After 24 hours, the amastigotes inside the monocyte cells were observed under inverted microscope.

Assays on L. chagasi promastigotes
Promastigotes were counted in a Neubauer chamber and used at a concentration of 1 × 10 5 promastigotes/well. On the first step, the oils of each plant were dissolved in ethanol, this solution was called the stock solution. On the second step, 100 µL of stock solution was diluted in milli-Q water add M199 medium for promastigotes or Dulbecco's medium for amastigotes. The oil solution was evaluated at 6.25, 12.5, 25, 50, and 100 µg.mL -1 , as previously described by Tempone et al. (2005). After a 24-h incubation with the relevant compound, a tetrazolium dye (MTT) colorimetric assay (bromide 3-4.5-dimethylthiazol-2-yl-2.5-dephenyltetrazolium) was performed on promastigotes to determine viability, and the IC50 of each treatment was calculated. The positive control for this assay was pentamidine, and the negative control was M199 medium. All oils were assayed in triplicate. The MTT assay was analyzed on a Multiskan MS (UNISCIENCE  ) microplate reader at a wavelength of 570 nm.

Assays on L. chagasi amastigotes
Plant oils were diluted in milli-Q water to 6.25, 12.5, 25, 50 and 100 µg.mL -1 and then added to microplates containing a confluent layer of cells and amastigotes. An in situ enzyme-linked immunosorbent assay (ELISA) to determine the IC50 was performed according to the method of Piazza et al. (1994). Prior to treatment, infected cells were stained, and parasites were observed by microscopy. Experiments were performed on cells exhibiting greater than 50% infection in a given microscope field. Briefly, after a 24 hours incubation with the indicated plant oil, a solution of 0.01% saponin (Sigma-Aldrich  ) and 1% bovine serum albumin (BSA; Sigma-Aldrich  ) in 1X phosphate-buffered saline (PBS) was added to each well, and plates were incubated for 30 minutes at 37 °C. Wells were subsequently blocked with 5% nonfat dry milk (Nestle  ) in PBS for 30 minutes at 37 °C. Microplates were washed and dried three times and then anti-L. chagasi serum, diluted 1:500 in 1X PBS containing 3% milk, 0.05% Tween 20 (PBSLT) and 10% FCS, was added, and plates were incubated at 37 °C overnight. Horseradish peroxidase-conjugated anti-rabbit IgG (Sigma-Aldrich  ) was diluted 1:10,000 in PBSLT and, after further washing, ortho-phenylenediamine chromogen (OPD) (Sigma-Aldrich  ) was added. To stop the reaction, 4 N chloric acid (Novaquímica  ) was added, and plates were read on a microplate reader using a 492 nm filter. The positive control for this assay was 40 µg.mL -1 amphotericin B (Sigma-Aldrich  ), and the negative control was Dulbecco medium alone. To generate anti-L. chagasi antibodies, rabbits were immunized with promastigotes, and serum was obtained 30 days after immunization.

Cytotoxicity assay
RAW 264.7 cells (Sigma-Aldrich  ) were grown in 96 well microplates and treated with 100 µg.mL -1 of plant oils. Subsequent testing was performed in situ by ELISA, as described above. The positive control in this step was 40 µg.mL -1 amphotericin B (Sigma-Aldrich  ), and the negative control was Dulbecco medium. Readings were performed using a 492 nm filter in a microplate reader.

Statistical analysis
The drug concentrations that achieved a 50% inhibition in the growth of parasites (IC50) were calculated using a nonlinear regression curve, with a 95% confidence interval. A one-way analysis of variance was the statistical method used to evaluate the response. Pair wise comparisons of means were made using Tukeys procedure. A p-value <0.05 was considered significant. For normalization, 100% survival was computed as the OD of the control containing only promastigotes or amastigotes, and/or cells alone.

Results
The results of oil activity on promastigotes showed that C. reticulata oleoresin and L. sidoides essential oil were effective compared with positive control pentamidine (p > 0.05). C. sativum essential oil was not effective against promastigotes of L. chagasi and was different from positive control (p < 0.05). The assay on amastigotes demonstrated that the three oils were effective, and they were not significantly different from amphotericin B (p > 0.05) ( Table 1).
The oleoresin from C. reticulata was not toxic to RAW 264.7 cells; the cells displayed 78.45% cell viability, which did not differ significantly from positive and negative controls (p > 0.05). In contrast, the other oils demonstrated some toxicity; 49.9% viability was observed in cells treated with C. sativum, and 57.8% viability was observed in cells treated with L. sidoides. These results were significantly different from the positive and negative controls and to oleoresin C. reticulata (p < 0.05) (Figure 1). Phytochemical analysis revealed the presence of terpenoids in the extracted oils. Further characterization determined that the principal constituents of L. sidoides essential oil, C. sativum essential oil and C. reticulata oleoresin were thymol (59.65%), β-linalool (73.21%) and β-caryophyllene (43.18%), respectively (Table 2).

Discussion
A study among eight plant species of the genus Copaifera (C. multijuga, C. officinalis, C. reticulata, C. lucens, C. langsdorffii, C. paupera, C. martii, and C. cearensis), related that C. reticulata demonstrated the greatest efficacy against Leishmania amazonensis, with IC50 values of 5 µg.mL -1 on promastigotes and 20 µg.mL -1 on amastigotes. This oil also exhibited a low level of toxicity toward the murine macrophage cell line J774G8 (SANTOS et al., 2008). The results showed that C. reticulata had IC50 values against promastigotes of L. chagasi similar to IC50 values on promastigotes of L. amazonensis found by Santos et al. (2008). However the oleoresin had proven to be even more effective against L. chagasi amastigotes (IC50 0.52 µg.mL -1 ). The differing IC50 values obtained with C. reticulata oleoresin on amastigotes of L. amazonensis and L. chagasi can be explained by differences in target. The potential targets of leishmanicidal drugs are glucose transport system, purines and other essential biomolecules and several important and specific enzyme systems of Leishmania (BARRETT et al., 1999;DOERIG et al., 2002;PADMANABHAN et al., 2005). Oliveira et al. (2009) had previously investigated the activity of essential oils from Cymbopogon citratus, L. sidoides and Ocimum gratissimum against promastigotes of L. chagasi. In this study, the most effective compound was C. citratus, with an IC50 of 45 µg.mL -1 , followed by the oil of O. gratissimum, with an IC50 of 75 µg.mL -1 ; the least effective essential oil was L. sidoides, with an IC50 of 89 µg.mL -1 . Another study performed on promastigotes and amastigotes of L. chagasi reported that essential oils from plants of the genus Lippia were effective in inhibiting promastigote infection, with IC50 values of 4.4 µg.mL -1 for L. origanoides, 5.2 µg.mL -1 for L. citriodora, 18.9 µg.mL -1 for L. alba, and 51.8 µg.mL -1 for L. micromera. No oil had demonstrated inhibition of intracellular amastigotes in the human monocyte line THP-1 (ESCOBAR et al., 2010). In contrast, in the present work, L. sidoides oil exhibited the greatest activity against both promastigotes and amastigotes of L. chagasi. The difference between the IC50 values may be due to the composition of the oils, which is highly variable, depending on the soil where the plant was grown, the time of collection, environmental factors and other effects (MARTINS et al., 2006;NOGUEIRA et al., 2007).
The effect of C. sativum essential oil against Leishmania parasites had never been reported, actually only one study was published with three fractions of C. sativum against Leishmania infantum (RONDON et al., 2011). In this study, the results against promastigotes and amastigotes were contradictory because all fractions had effect on promastigotes but the ethyl acetate fraction did not act on amastigotes. Among the results on promastigotes, the chloroform fractions demonstrated the higher IC50 value and only this fraction showed terpenoids in qualitative chemical analysis. Other studies reported the effect of C. sativum essential oil against microorganisms like Candida spp. (SILVA et al., 2011a), Gram-positive and Gram-negative bacteria (SILVA et al., 2011b).
The main constituents of C. sativum, L. sidoides and C. reticulata oils were the monoterpenes β-linalool and thymol, and the sesquiterpene β-caryophyllene, respectively. Similar findings were obtained by Ghannadi and Sadeh (1999) for C. sativum,  1. Effect of C. sativum and L. sidoides essentials oils, and C. reticulata oleoresin at a concentration of 100 µg.mL -1 , on the viability of the murine monocyte cell line, RAW 264.7. Amphotericin B (40 µg.mL -1 ) was used as a reference, and Dulbecco medium served as the negative control. *p < 0.05 1 . 1 The star symbol shows statistical difference between treatments 1 and 2 related to treatment 3 and the positive and negative controls (p < 0.05). The treatment 1 is not statistically different from treatment 2 (p > 0.05). Camurça-Vasconcelos et al. (2007) for L. sidoides and Santos et al. (2008) for C. reticulata. The mechanism of action of linalool found in the fruit of C. sativum is to stimulate the production of reactive oxygen species, such as nitric oxide (NO), to inhibit the activity of mitochondrial respiratory chain and to decrease levels of ATP and glutathione in the cells (USTA et al., 2009). Rosa et al. (2003) investigated the effects of linalool from essential oil of Croton cajucara against Leishmania amazonensis and on macrophage-parasite interactions. This compound had effect on both parasites forms and produced twice the amount of NO as the non treated macrophages. It reinforces the model that linalool acts indirectly to favor NO production by macrophages to eradicate the infection with Leishmania spp. C. sativum essential oil is rich in linalool. Such oil was more effective on amastigotes.
Thymol, a terpenoid, causes structural and functional damage to the cell membrane and acts directly on the synthesis and activity of ATPase and on the concentration of potassium and phosphate ions in the parasite. These activities have been characterized in bacteria, and this is the likely mechanism of action against Leishmania (LAMBERT et al., 2001;OSORIO et al., 2006). Synthetic thymol compounds exhibited an IC50 of 194.3 µg.mL -1 against promastigotes of Leishmania panamensis (ROBLEDO et al., 2005). In another study, a thymol-rich L. sidoides essential oil showed an IC50 of 89 µg.mL -1 against L. chagasi (OLIVEIRA et al., 2009). The essential oil of L. citriodora, which is also rich in thymol, demonstrated an IC50 of 4.4 µg.mL -1 on L. chagasi promastigotes (ESCOBAR et al., 2010). In this report, the essential oil of L. sidoides proved to be rich in thymol and yielded an IC50 of 19.76 µg.mL -1 on promastigotes of the same species. Synthetic thymol exhibited higher IC50 values than the essential oils, which are composed of natural thymol and other substances. Thus, the more effective action of the oils may be due to the synergistic effect of other compounds, such as caryophyllene and cymene, or perhaps thymol is not as potent against L. panamensis compared to L. chagasi.
While the mechanism of action of β-caryophyllene is not well understood, it is effective against L. amazonensis (SANTOS et al., 2008) and exhibits anti-inflammatory (SHIMIZU et al., 1990), antimicrobial and antioxidant properties (SAHIN et al., 2004).
Investigations using natural products have shown great potential in finding new agents to fight tropical diseases, and the results here demonstrate that C. reticulata oleoresin is highly effective against promastigotes and amastigotes of L. chagasi, while exhibiting no toxicity toward the RAW 264.7 cell line. While further research is needed to prove its efficacy in vivo, C. reticulata oleoresin may be a promising new treatment for canine and human visceral leishmaniasis.