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In vitro cytocidal effects of the essential oil from Croton cajucara (red sacaca) and its major constituent 7- hydroxycalamenene against Leishmania chagasi
- Igor A Rodrigues†1, 2,
- Mariana M B Azevedo†3,
- Francisco C M Chaves4,
- Humberto R Bizzo5,
- Suzana Corte-Real6,
- Daniela S Alviano1,
- Celuta S Alviano1,
- Maria S S Rosa1 and
- Alane B Vermelho1Email author
© Rodrigues et al.; licensee BioMed Central Ltd. 2013
Received: 29 March 2013
Accepted: 1 October 2013
Published: 2 October 2013
Visceral leishmaniasis is the most serious form of leishmaniasis and can be lethal if left untreated. Currently available treatments for these parasitic diseases are frequently associated to severe side effects. The leaves of Croton cajucara are used as an infusion in popular medicine to combat several diseases. Previous studies have demonstrated that the linalool-rich essential oil from C. cajucara (white sacaca) is extremely efficient against the tegumentary specie Leishmania amazonensis. In this study, we investigated the effects of the 7-hydroxycalamenene-rich essential oil from the leaves of C. cajucara (red sacaca) against Leishmania chagasi, as well as on the interaction of these parasites with host cells.
Promastigotes were treated with different concentrations of the essential oil for determination of its minimum inhibitory concentration (MIC). In addition, the effects of the essential oil on parasite ultrastructure were analyzed by transmission electron microscopy. To evaluate its efficacy against infected cells, mouse peritoneal macrophages infected with L. chagasi promastigotes were treated with the inhibitory and sub-inhibitory concentrations of the essential oil.
The minimum inhibitory concentrations of the essential oil and its purified component 7-hydroxycalamenene against L. chagasi were 250 and 15.6 μg/mL, respectively. Transmission electron microscopy analysis revealed important nuclear and kinetoplastic alterations in L. chagasi promastigotes. Pre-treatment of macrophages and parasites with the essential oil reduced parasite/macrophage interaction by 52.8%, while it increased the production of nitric oxide by L. chagasi-infected macrophages by 80%.
These results indicate that the 7-hydroxycalamenene-rich essential oil from C. cajucara is a promising source of leishmanicidal compounds.
Human visceral leishmaniasis (HVL) or kala-azar is an often lethal infectious disease. About 500,000 new cases of visceral leishmaniasis are reported worldwide each year . In Brazil, approximately 4,000 people are infected with leishmaniasis each year, and 10.5% die from the disease. The disease is more common in the Northeastern part of the country, but it extends to tropical forest regions and to some major industrial cities in the Southeastern region [2, 3].
Conventional chemotherapy, one of the most common treatments for leishmaniasis, is highly toxic and fails in approximately 10% of cases . Among the chemotherapeutic agents used to treat the disease, the pentavalent antimonials are still the first choice. However, the current scenario of drug development for leishmaniasis is more promising than a few decades ago. Recently, potential therapies for visceral leishmaniasis have been introduced, including liposomal amphotericin B, paromomycin, and miltefosine . Despite the advances, both the conventional treatments and the new chemotherapeutic agents have a number of important disadvantages such as severe side effects and high cost.
Prompted by the fact that the essential oils extracted from leaves of white and red sacaca present antimicrobial properties, being effective against several microorganisms, including L. amazonensis, Staphylococcus aureus MRSA, Mycobacterium smegmatis, M. tuberculosis, Rhizopus oryzae, and Candida albicans[5–7], we decided to investigate the effects of the 7-hydroxycalamenene-rich essential oil of red sacaca against Leishmania chagasi parasites. In addition, the effects of the essential oil on the interaction of these parasites with mammalian host cells were evaluated.
Culture media were purchased from Difco (Sparks, MD 21152, USA). Reagents used in electrophoresis and molecular mass standards were acquired from Amersham Life Science (Little Chalfont, England). All other reagents were analytical grade.
Plant material, essential oil extraction and 7-hydroxycalamenene purification
All samples were kept in a germplasm bank under the same cultivation practices. Leaves of C. cajucara were collected between 08:00 and 09:00 AM. Voucher specimens were deposited at the Embrapa Occidental Amazon Herbarium (registry IAN 165013). The oils were obtained by hydrodistillation in a modified Clevenger apparatus for 4 hours, carefully separated, and stored in opaque glass vials in a refrigerator (−10°C) prior to analysis and biological assays .
The isolation of 7-hydroxycalamenene was performed by preparative column chromatography on silica gel (Merck, 70–230 mesh), eluting with hexane and hexane-ethyl acetate mixtures.
Analysis of the essential oil by GC-MS
The essential oils were analyzed at GC-MS under the following conditions: the oven temperature was programmed from 60°C to 240°C at 3°C/min, and helium was the carrier gas (at 1.0 mL/min). One microliter of 1% solution of the oil in dichloromethane was injected in split mode (1:100). Mass spectra were obtained in an Agilent 5973N system, fitted with a low bleeding 5% phenyl/95% methylsilicone (HP-5 MS, 30 m × 0.25 mm × 0.25 μm) fused silica capillary column, operating in electronic ionization mode (EI) at 70 eV, with scan mass range of 40–500 m/z. Sampling rate was 3.15 scan/s. Ion source was kept at 230°C, mass analyzer at 150°C, and transfer line at 260°C. Linear retention indices (LRI) were measured by injection of a series of n-alkanes (C7-C30) in the same column and conditions as described above and compared with reference data .
Analyses of 7-hydroxycalamenene by GC
In order to evaluate the degree of purity of the isolated material, 7-hydroxycalamenene was analyzed on an Agilent (Palo Alto, CA, USA) 6890N gas chromatograph fitted with a 5% phenyl—95% methylsilicone (HP-5, 30 m × 0.32 mm × 0.25 μm) fused silica capillary column. The oven temperature was programmed from 60°C to 240°C (3°C/min), and hydrogen was used as carrier gas (1.4 mL/min). It was injected 1.0 μL of a 1% solution of the 7-hydroxycalamenene in dichloromethane, in split mode (1:100). Injector was kept at 250°C and detector (FID) at 280°C. All analyses were performed in triplicate.
The percentage was calculated as % peak area of GC–FID. The material purity was over 80%.
Promastigote forms of Leishmania (L.) chagasi MHOM/BR/1974/PP75 were obtained from Leishmania Type Culture Collection (LTCC) of Oswaldo Cruz Institute/Fiocruz (Rio de Janeiro, RJ, Brazil). Parasites were maintained by weekly transfers in PBHIL medium supplemented with 10% fetal bovine serum (FBS), at 28°C . Parasites were maintained infective by periodical macrophage infection.
To evaluate the minimum growth-inhibitory concentration (MIC), promastigote forms of L. chagasi (106 parasites/mL) were incubated in fresh medium in the presence of several concentrations (1–1000 μg/mL) of essential oil and its 7-hydroxycalamenene-rich purified fraction (80%). After 120 h incubation, parasite viability was determined using the microplate method based on the reduction of resazurin as described . Alternatively, cells were centrifuged and washed three times in PBS (150 mM NaCl; 20 mM phosphate buffer, pH 7.2) and resuspended in fresh culture medium without the plant extract, to evaluate the leishmanicidal or leishmanistatic effect. The 50% lethal dose (LD50) was determined by logarithmic regression analysis of the data obtained as described above.
Transmission electron microscopy
Parasites at early stationary phase of growth were harvested, washed twice with PBS, and incubated or not with 250 μg/mL (MIC concentration) of the essential oil at 28°C for 24 hours. Cells were washed twice in PBS and then fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer containing 3.5% sucrose, pH 7.4, at 4°C for 60 min. Parasites were then rinsed in PBS pH 7.4 and pelleted by centrifugation. The pellets were then post-fixed with a 1% osmium tetroxide and potassium ferrocyanide solution for 1 hour, dehydrated sequentially in acetone, and then embedded in Epon 812. Ultrathin sections were cut using an LKB ultramicrotome and collected on copper grids. Sections were stained with uranyl acetate and lead citrate and were observed in a Jeol JEM1011 transmission electron microscope.
Peptidase activity of Leishmania chagasi by gelatin–SDS-PAGE
Promastigote forms of L. chagasi (2.0x106 parasites/mL) were harvested at the log phase of growth, washed two times in PBS by centrifugation, and then incubated in PBS pH 6.8 with the essential oil (250 μg/mL) at 28°C for 1 hour. Controls were prepared without the essential oil. Cells were centrifuged and the supernatants collected and concentrated by dialysis (cut-off 9000 Da) against polyethylene glycol 4000 for 12 hours at 4°C. Cells were disrupted at 4°C by four 15-s periods of ultrasound treatment with 1-min intervals. The extracts were centrifuged and supernatant aliquots were stored at −60°C. Polyacrylamide gels containing 0.2% copolymerized gelatin as substrate were loaded with 28 μg of proteins (cellular extract and supernatants) per slot. After electrophoresis at a constant voltage of 200 V at 4°C, gels were soaked in 2.5% Triton X-100 (1 h) to remove SDS, and then incubated for 18 h at 37°C in 0.1 M phosphate buffer, pH 5.5, in the absence or in the presence of protease inhibitors (1 mM E-64 and 1 mM phenanthroline). Gels were stained for 1 h with 0.2% Comassie brilliant blue R-250 in methanolacetic acid-water (50:10:40) and de-stained to expose proteolytic bands in the same solvent. The relative molecular mass of the peptidases was calculated by comparison with the mobility of low molecular mass standards. Peptidase activity was estimated by the intensity of the bands using ImageJ program (NIH).
Peritoneal mouse macrophages and cytotoxity assay
Female Swiss mice (6–8 weeks old) from a colony at the General Microbiology Department/UFRJ animal house facility were used in all of the experiments. The animals were maintained at 21 ± 2°C, on a 12hs light/dark cycle, with food and water until 1 h prior to the experimental procedures. The animals were killed according to the federal guidelines and institutional policies by cervical dislocation. The procedures were approved by the Fiocruz Commitee of Ethics for the Use of Animals (resolution 242/99, license LW 2/12). Peritoneal mouse macrophages were collected in cold PBS and allowed to adhere onto coverslips placed in 24-well culture plates, for 30 min at 37°C and 4% CO2. For the cytotoxity assay, 105 macrophages/well were incubated with different concentrations of the C. cajucara essential oil (1–1000 μg/mL) at 37°C and 5% CO2 for 48 h. Cell viability was assessed after 4 h incubation with resazurin as described by Al-Musayeib et al..
Macrophage infection and NO production
Parasites and/or peritoneal mouse macrophages were pretreated with 250 and 125 μg/ml (MIC and subMIC) of red sacaca essential oil. After 20 min, adherent cultured macrophages and free parasites were washed once, resuspended in fresh RPMI culture medium, and then co-cultured at a ratio of 10 promastigotes to 1 macrophage at 37°C for 120 min in a 4% CO2 atmosphere. Cells were fixed and Giemsa stained, and the percentage of infected macrophages was determined by counting 600 cells. The association indices were determined by multiplying the percentage of infected macrophages by the mean number of parasites per infected cell. The association index was considered as the number of parasites that successfully infected macrophages. Nitrite levels in culture supernatants of macrophages infected or not with L. chagasi were analyzed by Griess reaction as previously described . The absorbance at 550 nm was measured, and the concentration of nitrite was calculated using a linear regression of a standard curve.
Anti-intracellular amastigote activity
The effects of the essential oil from red sacaca (250 and 125 μg/mL) on intracellular amastigotes were determined after treatment of pre-infected macrophages as previously described with slight modifications . Briefly, mouse peritoneal macrophages were infected with L. chagasi promastigotes (logarithmic growth phase) as described above. Next, free promastigotes were removed by extensive washing with PBS and the infected macrophages were then incubated for 24 h to allow complete promastigote differentiation into intracellular amastigotes. Then, MIC and subMIC concentrations of the essential oil were added to the cultures of infected macrophages for 20 min. After treatment, supernatants were collected for the analysis of NO production and coverslips were fixed as previously described above.
All experiments were performed in triplicate. All results are presented as the mean and standard error of the mean (SEM). Normalized data were analyzed by one-way analysis of variance (ANOVA), and differences between groups were assessed using the Student-Newman-Keuls post-test. Results were considered significant at p ≤ 0.05.
Results and discussion
The crucial role that traditional medicine plays in health care of people living in developing countries is recognized worldwide. For centuries, traditional medicine was the only health care system available for the prevention and treatment of several diseases in different cultures . The research on natural products around the world has found literally thousands of phytocompounds that are biologically active against various a number of illnesses, including infectious diseases. In this study, we described the cytocidal activity of the 7-hydroxycalamenene-rich essential oil from C. cajucara, red sacaca, against the etiological agent of Kala-azar, L. chagasi.
In vitro activity of C. cajucara (red sacaca) essential oil and its purified 7-hydroxycalamenene-rich fraction against L. chagasi promastigotes and toxicity profile in mouse peritoneal macrophages
7-hydroxycalamenene purified fraction
Most of the conventional antileishmanial drugs exhibit strong in vitro activity against the parasites but they are also highly toxic for mammalian host cells. By contrast, several studies have demonstrated that crude essential oils and their major compounds present low or no toxicity to host cells at the effective concentrations [27–30]. Both the essential oil and its 7-hydroxycalamenene-rich purified fraction presented no toxicity for peritoneal mouse macrophages at the concentrations used in this study.
Our results further support the antileishmanial activity of 7-hydroxycalamenene-rich essential oil of C. cajucara (red sacaca) leaf extracts. Its toxicity against L. chagasi, with no effect on mammalian cells, indicates that this essential oil is a promising source of antileishmanial agents. Further studies, including in vivo bioassays, are needed to validate the in vitro results and to ascertain the safety of the essential oil and the 7-hydroxycalamenene purified fraction.
The authors thank Flavia Nascimento, Naiany Alves de Jesus, and Denise da Rocha de Souza for their technical support. They also thank Plataforma Rudof Barth of Instituto Oswaldo Cruz (Fiocruz/RJ) for the acquisition of the electron micrographs. This work was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico (MCT/CNPq), Coordenação de Aperfeiçoamento Pessoal de Nível Superior (CAPES), and Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ).
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