Abstract
Context: Leishmaniasis is a widespread tropical infection caused by different species of Leishmania protozoa. There is no immunoprophylaxis (vaccination) available for Leishmania infections and conventional treatments are unsatisfactory; therefore antileishmanial drugs are urgently needed. Natural products are attractive due to their structural diversity.
Objective: The present work investigated the antileishmanial action of 21 species of plants.
Materials and methods: Plants were collected and their hydroalcoholic extracts were screened against promastigotes and amastigotes of L. amazonensis. Their toxicity was also assayed against peritoneal macrophages from BALB/c mice.
Results: Five extracts showed significant growth inhibitory activity against promastigote form. Only the extracts from Bidens pilosa L. (Asteraceae) and Punica granatum L. (Punicaceae) inhibited the growth of intracellular amastigotes, with IC50 values of 42.6 and 69.6 µg/mL, respectively. In addition, a low toxicity on macrophage from BALB/c mice was observed.
Discussion: The antiparasitic activities of B. pilosa and P. granatum have been reported against other parasitic agents and their actions can be the results of flavonoids present in the extracts.
Conclusion: This study supports the importance of natural products as potential sources in the search for new antileishmanial drugs.
Introduction
Leishmaniasis comprises a group of infectious diseases caused by hemoflagellate organisms of the genus Leishmania, which are obligate intracellular parasites of mononuclear phagocytes of vertebrate hosts (CitationAlexander & Russell, 1992). Nowadays, leishmaniasis is prevalent in 88 tropical countries throughout the world and an estimate of 1.5 million to 2 million new cases occur annually. Moreover, approximately 350 million people live at risk of infection with Leishmania parasites. The World Health Organization (WHO) has identified leishmaniasis as a major public health problem, particularly in Latin America (CitationVidyashankar & Noel, 2002).
Control of leishmaniasis relies on chemotherapy, but the availability and effect of conventional drugs are limited. Pentavalent antimonials: sodium stibogluconate (Pentostam) and N-methylglucamine (glucantime) have been the first line of antileishmanial defense for many years, but they have been rendered obsolete in several Old World regions by the emergence of antimony resistance. Treatment failure has also been reported in the New World (CitationSundar, 2001). Alternative treatments include the polyene antibiotic amphotericin B, the aminoglycoside paramomycin, the alkylphospholipid miltefosine, and the azole ketoconazole (CitationSingh & Sivakumar, 2004). These drugs are toxic, expensive and require long duration for therapy. Given the prospect that antileishmanial vaccines are only under clinical assays (CitationKhamesipour et al., 2006), the search for better drugs, more effective, less toxic and easier to use is urgently needed (CitationCroft et al., 2006).
Natural products may offer an unlimited source of chemical diversity for identification of new drugs. In endemic areas (CitationChan-Bacab & Peña-Rodríguez, 2001), a number of traditional plants are commonly used to treat infectious diseases. The Program of Tropical Diseases of WHO considered an investigation of any plant as essential priority for the treatment of Leishmania. In the present work, the antileishmanial activities of 21 plants growing in Cuba are evaluated against promastigotes and amastigotes of L. amazonensis, as well as their cytotoxicity against peritoneal macrophage from BALB/c mice. The plants were selected due to their popular use as antiparasitic and easy cultivation of samples.
Methods
Plant materials
Vegetative samples of 21 species () were collected in May 2003, in Havana City. All plants were collected and authenticated by Ramón Scull (Institute of Pharmacy and Food) according to the Cuban Flora. Their voucher specimen or collector’s numbers were assigned and a sample of each was deposited in the herbarium of the National Botanical Garden (NBG), Havana City, Cuba.
Table 1. Alphabetical list of 21 plants growing in Cuba screened in the study and antiparasitic studies reported.
Preparation of plant extracts
The hydroalcoholic extracts were prepared as previously described (CitationRodríguez et al., 2006). Briefly, the leaves of all samples () and seeds of Bixa orellana were dried in an oven with ventilation system at 30°C and crushed. The fluid extracts were prepared by maceration for seven days using ethanol 80% as solvent (CitationRamal, 1992). The extracts were evaporated, lyophilized and dissolved in ethanol 80% at 20 mg/mL.
Reference drug
Pentamidine (Richet, Buenos Aires, Argentina) was used as positive control; using as stock solution a concentration of 10 mg/mL.
Parasites
The MHOM/77BR/LTB0016 strain of Leishmania amazonensis was kindly provided by the Department of Immunology, Oswaldo Cruz Foundation (FIOCRUZ), Brazil. Parasites were routinely isolated from mouse lesions and maintained as promastigotes at 26°C in Schneider’s medium (Sigma, St. Louis, MO, USA) containing 10% heat-inactivated fetal bovine serum (HFBS) (Sigma), 100 μg of streptomycin/mL, and 100 U of penicillin/mL.
Antipromastigote screening
Growth inhibition of L. amazonensis promastigotes was evaluated at final concentrations of 50 and 100 μg/mL, which are used in studies about screening of different plant extracts against Leishmania (CitationPlock et al., 2001). The extracts were added to cultures at 105 promastigotes/mL. After 72 h of incubation, parasites were incubated for 3 h with p-nitrophenyl phosphate (20 mg/mL) dissolved in 1 M sodium acetate buffer (BDH, Poole, Dorset), pH 5.5, with 1% Triton X-100 (BDH) at 37°C. The absorbance was determined in an EMS Reader MF version 2.4-0, at a wavelength of 405 nm (CitationBodley et al., 1995; CitationMontalvo et al., 2000). Results were expressed as a percentage of inhibition growth (%I) in comparison to controls treated with the solvent (H2O:EtOH, 20:80, v:v) at the same concentration. All evaluations were performed in triplicate and standard deviation was calculated.
Antiamastigote activity
Resident macrophages were collected from peritoneal cavities of healthy BALB/c mice on RPMI 1640 medium (Sigma) supplemented with antibiotics, plated at 106/mL in 24-well Lab-Tek (Costar®, USA) and left to adhere for 2 h at 37°C in 5% CO2. Non-adherent cells were removed by washing with PBS. Stationary-phase L. amazonensis promastigotes were added at a 4:1 parasite/macrophage ratio. Cultures were added for a further 4 h and cell monolayers were washed to remove free parasites. Then, 1990 μL of the RPMI complete medium and 10 μL of the different extracts were added, following serial dilutions 1:2, to obtain final concentrations between 12.5 to 100 μg/ mL, and incubated for a further 48 h (CitationCaio et al., 1999). Cultures as control were included, which were treated with the solvent at the same concentration. Cultures were then fixed with absolute methanol, stained with Giemsa, and examined under light microscopy. The number of intracellular amastigotes were determined in 100 macrophages per each sample. Results were expressed as percentage of reduction of the infection rate (%IR) in comparison with those obtained with positive controls. The infection rates were obtained by multiplying the percentage of infected macrophages by the number of amastigotes per infected macrophages (CitationDelorenzi et al., 2001). The median inhibitory concentration (IC50) value was calculated by linear regression analysis. Each experiment was performed twice on different occasions with two replicates for each one. The results were expressed as their average and standard deviation (SD).
Cytotoxicity assay
We determined the IC50 of the extracts on peritoneal macrophage from BALB/c mice. Macrophages were collected and cultured at 105 cell/mL. After 2 h of incubation at 37°C in 5% CO2, non-adherent cells were removed and 2 μL of extract dilutions, previously prepared in medium, were added in 198 μL of medium with 10% HFBS and antibiotics. Macrophages were treated with the extracts from 1.5 to 200 μg/mL for 48 h. Cultures as negative control were included, which were treated with the solvent at the same concentration. The cytotoxicity was determined using the colorimetric assay with 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (Sigma). MTT solutions (15 μL) were added to each well at 5 mg/mL in PBS. After incubation for an additional 4 h, the medium was eliminated and formazan crystals were dissolved by addition of 100 μL of DMSO. Absorbance was determined using an EMS reader MF Version 2.4-0, at a wavelength of 560 nm (CitationSladowski et al., 1993). The IC50 was obtained from dose-response curves fit to data by means of the equation for the sigmoidal Emax model. This test was performed in triplicate and the results were expressed as their average and standard deviation.
The selectivity index (SI) ratio (IC50 for macrophage/IC50 for amastigotes) was used to compare the toxicity of the extracts for murine macrophage and the activity against intracellular amastigotes of Leishmania. SI >1 is considered more selective for activity against parasites, and SI <1 is considered more selective for activity against cells (CitationShioji et al., 2005).
Results
lists the 21 plant species screened in this study. The given information includes: plant families, local names and their studies about antiparasitic effects. Bibliography about the pharmacological activity of Cassia grandis L.f. (Caesalpiniaceae), Cupressus sempervirens L. (Cupressaceae), Pluchea carolinensis (Jacq.) G. Don (Asteraceae) was not found against parasitic diseases, but there exist some reports about their protective role against bacteria, fungi and viruses.
The hydroalcoholic extracts were used in order to assay their antileishmanial effect. shows the activity of the extracts against L. amazonensis promastigotes at 50 and 100 μg/mL. The addition of 100 μg/mL to Bidens pilosa, Bixa orellana, Melia azedarach and Parthenium hysterophorus caused an inhibition of more than 50% on parasite growth after 72 h. The extract of Punica granatum at 100 μg/mL showed inhibition proximity of 50%. The five plants previously mentioned, were selected to perform the studies against intracellular amastigotes.
Table 2. Inhibition growth of promastigotes of L. amazonensis incubated with plant extracts and reference drug (pentamidine).
The treatment of murine macrophages infected with L. amazonensis showed that all extracts inhibited the growing of the parasite (). In addition, uninfected murine macrophages were also treated to determine safety and to calculate the SI. The extracts showing most promising activity were P. granatum and B. pilosa, with an SI ratio of 4.
Table 3. Antileishmanial activity (IC50), cytotoxicity (IC50) and selectivity (SI) of plant extracts and reference drug (pentamidine).
P. hysterophorus and B. orellana extracts gave the best IC50, with values of 20.2 and 26.8 μg/mL, respectively. However, these extracts caused a high toxicity, with IC50 values of 37.8 and 60.6 µg/mL, respectively. M. azedarach extract showed more activity for mammalian cells than the parasites (SI <1).
Discussion
The aim of this study was to search for the antileishmanial activity of some plants growing in Cuba that displayed antimicrobial activity. Seventeen plants have been studied previously against parasitic diseases as summarized in . The most common reports are against Plasmodium (CitationAntoun et al., 2001; CitationAndrade-Neto et al., 2004; CitationRodríguez et al., 2006; CitationKvist et al., 2006) and Trypanosoma (CitationGonzalez-Coloma et al., 2002; CitationTalakal et al., 1995; CitationWurochekke & Nok, 2004) parasites. We did not find antiparasitic reports for three plants. The selected plants are abundant in the wild and their cultivation is easy with a low cost, which guarantees enough quantities to perform the pharmacological evaluations.
In the case of Leishmania, the screening is usually done on promastigote cultures, which is easy, reproducible and quick. However, the promastigotes are not the infective form of the parasite in vertebrate hosts. So this preliminary evaluation must be complemented with an evaluation using intracellular amastigotes in macrophages. At the same time, an evaluation of the possible cytotoxicity of the products must be carried out using non-parasitized macrophages. We compared the activity of selected extracts against intracellular amastigotes and the cytotoxicity displayed against peritoneal macrophage, which establish if in vitro activity of the extract is due to its general cytotoxicity activity or if it possesses a selective activity against Leishmania (CitationChan-Bacab & Peña-Rodríguez, 2001; CitationCroft et al., 2006). The preliminary screening of herbal products showed that 81% of the tested plants had at least a 50% leishmanicidal activity at 100 μg/mL. Among the plant species evaluated here, B. pilosa caused an 85% of inhibition of promastigotes growth. Against amastigotes, an IC50 value of 43 μg/mL was obtained. This extract had a SI of 4, which demonstrates the presence of compounds with reasonable potency.
Bidens pilosa is an annual plant from tropical America. The ethanol extract was proven to be active against Plasmodium falciparum drug-resistant parasites in vitro and in rodent malaria in vivo (CitationAndrade-Neto et al., 2004), which constitutes a previous demonstration of its antiparasitic action. However, this plant is claimed to be useful for immune or anti-inflammatory disorders, specifically inhibiting TH1 cell differentiation (CitationChang et al., 2005). This immunomodulatory action will be negative to leishmaniasis control, as Leishmania is an intracellular obligate parasite, and the induction of TH1 response from mammalian host is needed to protect against infection (CitationAlexander & Russell, 1992). For that reason, an evaluation on an experimental model of cutaneous leishmaniasis could give more details about the efficacy of the extract from B. pilosa.
On the other hand, P. granatum showed less activity, but the SI was the most relevant value, which indicated a major selectivity of the extract. CitationCalzada et al. (2006) reported that a methanol extract of P. granatum was active against Entamoeba histolytica and Giardia lamblia. Other studies have reported the activity of this plant for antiviral actions, which demonstrated that the activity of P. granatum is caused by complex blocking of virus binding to CD4 cell and inhibiting the infection (CitationNeurath et al., 2004). This action can suggest the antileishmanial effect of this plant, due to Leishmania being an intracellular parasite that requires the binding to macrophages as a prerequisite to infection.
The antiparasitic activity of B. pilosa and P. granatum can be the result of flavonoids present in the ethanolic extracts, which have been demonstrated in both plant extracts (CitationAndrade-Neto et al., 2004; CitationSudheesh & Vijayalakshmi, 2005).
Bixa orellana and P. hysterophorus showed the best activity against both promastigote and amastigote forms. However, the toxicity was high against macrophage, which indicates an unspecific action, due to the small values of SI obtained. Melia azedarach showed an IC50 against macrophages smaller than the IC50 value for amastigote; which indicates that the extract displayed a major toxicity compared with the antileishmanial activity. This case illustrates one of the major difficulties in pursuing natural products, because many compounds in the plant are produced as a biological defense mechanism (CitationPink et al., 2005). In general, the SI values were low and the IC50 against amastigotes were high, but the results obtained can be validated in studies on animal models. Differences in in vitro and in vivo situations can be found due to a multicellular organism metabolic transformation of compounds which may occur.
The validation of medicinal plants is needed. Since no local leishmaniasis exist in our country, the results of the present screening should be interpreted as a preliminary search of potential source of bioactive agents. The biodiversity of Cuban flora constitutes a new approach to identify relevant antileishmanial candidates. Previous studies evaluating the potential of plants growing in Cuba have been reported. For example, the essential oil from Chenopodium ambrosioides (Dysphania ambrosioides) showed a potent activity in vitro and in vivo against L. amazonensis (CitationMonzote et al., 2006).
Surveying the literature on antileishmanial pharmacological reports, we found that extracts from Artemisia absinthium (CitationPlock et al., 2001) and Mangifera indica (CitationKvist et al., 2006) have been previously evaluated against Leishmania parasites. We corroborate that these extracts do not show a relevant antileishmanial activity. On the other hand, an essential oil extracted from Ocimum sanctum showed in vitro activity against Leishmania donovani (CitationZheljazkov et al., 2008) and curcumin, the active principle of Curcuma longa, was capable of blocking the action of both nitric oxide and nitric oxide congeners on the Leishmania parasite (CitationChang et al., 2005). However, we did not observe a relevant antileishmanial activity with these extracts tested in our study.
In vitro studies showed that all extracts evaluated were less active than pentamidine, which is a drug used clinically, and their activity has been demonstrated against the promastigote and amastigote forms of parasite. However, these extracts are complex mixtures of substances and the active principles can be in a low concentration. Further investigations should be performed in order to fractionate and identify their active components.
Conclusion
In conclusion, considering that leishmaniasis constitutes a serious public health problem, the search for new drugs with high activity and reduced adverse effects deserves more effort. The search for new antileishmanial agents from plants can be a good alternative due to their high chemical diversity and a long history of use in humans. We think that the antileishmanial activity shown by crude extracts from B. pilosa and P. granatum, as well as the toxicity effect observed, justify the continuation of their study as a potential source of new drugs against Leishmania. The purification and identification of their active principles can be to performed. Additionally, the screening of antileishmanial properties of other plants growing in Cuba could be explored.
Acknowledgment
Thanks to Sheila Cabezas for helpful advice and revision of the paper.
Declaration of interest
The authors report no conflict of interest and have received no payment in preparation of this manuscript.
References
- Alexander J, Russell DG (1992): The interaction of Leishmania species with macrophages. Adv Parasitol 31: 175–254.
- Andrade-Neto VF, Brandão MG, Oliveira FQ, Casali VW, Njaine B, Zalis MG (2004): Antimalarial activity of Bidens pilosa L. (Asteraceae) ethanol extracts from wild plants collected in various localities or plants cultivated in humus soil. Phytother Res 18: 634–639.
- Antoun MD, Ramos Z, Vazques J, Oquendo I, Proctor GR, Gerena L, Franzblau SG (2001): Evaluation of the flora of Puerto Rico for in vitro antiplasmodial and antimycobacterial activities. Phytother Res 15: 638–642.
- Asha MK, Prashanth D, Murali B, Padmaja R, Amit A (2001): Antihelmintic activity of essential oil of Ocimum sanctum and eugenol. Fitoterapia 72: 669–670.
- Bodley AL, McGarry MW, Shapiro TA (1995): Drug cytotoxicity assay for African trypanosomes and Leishmania species. J Infect Dis 172: 1157–1159.
- Caio E, Lima D, Kaplan MAC, Nazareth M, Rossi-Bergmann B (1999): Selective effect of 2,6-dihydroxy-4methoxychalcone isolated from Piper aduncum on Leishmania amazonensis. Antimicrob Agent Chemother 43: 1234–1241.
- Calzada F, Barbosa E, Cedillo-Rivera R (2003): Antiamoebic activity of benzyl glucosinolate from Lepidium virginicum. Phytother Res 17: 618–619.
- Calzada F, Yepez-Mulia L, Aguilar A (2006): In vitro susceptibility of Entamoeba histolytica and Giardia lamblia to plants used in Mexican traditional medicine for the treatment of gastrointestinal disorders. J Ethnopharmacol 108: 367–370.
- Chan-Bacab MJ, Peña-Rodríguez LM (2001): Plant natural products with leishmanicidal activity. Nat Prod Rep 18: 674–688.
- Chang CL, Kuo HK, Chang SL, Chiang YM, Lee TH, Wu WM (2005): The distinct effects of a butanol fraction of Bidens pilosa plant extract on the development of Th1-mediated diabetes and Th2-mediated airway inflammation in mice. J Biomed Sci 12: 79–89.
- Croft S, Seifert K, Yardley V (2006): Current scenario of drug development for leishmaniasis. Indian J Med Res 123: 399-410.
- Delorenzi JC, Attias M, Gattass CR, Andrade M, Rezende C, Da Cunha A (2001): Antileishmanial activity of an indole alkaloid from Peschiera australis. Antimicrob Agent Chemother 45: 1349–1354.
- Gonzalez-Coloma A, Guadaño A, Inés C, Martínez R, Cortés D (2002): Selective action of acetogenin mitochondrial complex I inhibitors. Naturforsch 57c: 1028–1034.
- Hordegen P, Hertzberg H, Heilmann J, Langhans W, Maurer V (2003): The anthelmintic efficacy of five plant products against gastrointestinal trichostrongylids in artificially infected lambs. Vet Parasitol 117: 51–60.
- Irobi ON, Moo-Young M, Anderson WA (1996): In vitro clonal propagation of annatto (Bixa orellana L.). Pharm Biol 34: 87–90.
- Khamesipour A, Rafati S, Davoudi N, Maboudi F, Modabber F (2006): Leishmaniasis vaccine candidates for development: A global overview. Indian J Med Res 123: 423–438.
- Kvist LP, Christensen SB, Rasmussen HB, Mejia K, Gonzalez A (2006): Identification and evaluation of Peruvian plants used to treat malaria and leishmaniasis. J Ethnopharmacol 106: 390–402.
- Lans C, Brown G (1998): Ethnoveterinary medicines used for ruminants in Trinidad and Tobago. Prevent Vet Med 35: 149–163.
- Lans C, Harper T, Georges K, Bridgewater E (2000): Medicinal plants used for dogs in Trinidad and Tobago. Prev Vet Med 45: 201–220.
- Lans C, Harper T, Georges K, Bridgewater E (2001): Medicinal and ethnoveterinary remedies of hunters in Trinidad. BMC Complement Alternat Med 1: 10.
- Mesia GK, Tona GL, Nanga TH, Cimanga RK, Apers S, Cos P, Maes L, Pieters L, Vlietinck AJ (2008): Antiprotozoal and cytotoxic screening of 45 plant extracts from Democratic Republic of Congo. J Ethnopharmacol 115: 409–415.
- Montalvo AM, Sifontes S, Montano I, Espino AM (2000): [Need to use chromate to quantifier promastigotes of Leishmania in plates of 96 wells]. Rev Cub Med Trop 52: 95–100.
- Monzote L, Almannoni S, Montalvo AM, Scull R, Miranda M, Abreu J (2006): Activity of essential oil from Chenopodium ambrosioides grown in Cuba against Leishmania amazonensis. Chemotherapy 52: 130–136.
- Neurath AR, Strick N, Li YY, Debnath AK (2004): Punica granatum (pomegranate) juice provides an HIV-1 entry inhibitor and candidate topical microbicide. BMC Infect Dis 4: 41.
- Ramal N (1992): Norma Ramal de Salúd pública 309: [Regulation Norm of Public Health 309: Medicament from vegetal source. Crude drugs. Methods of assays]. Habana, Ministerio de Salud Pública, pp. 16–29.
- Pink R, Hudson A, Mouriès MA, Bendig M (2005): Opportunities and challenges in antiparasitic drug discovery. Nature Rev 4: 727–740.
- Plock A, Sokolowska-Köhler W, Presber W (2001): Application of flow cytometry and microscopical methods to characterise the effect of herbal drugs on Leishmania spp. Exp Parasitol 97: 141–153.
- Quinlan MB, Quinlan RJ, Nolan JM (2002): Ethnophysiology and herbal treatments of intestinal worms in Dominica, West Indies. J Ethnopharmacol 80: 75–83.
- Rodríguez M, Mendiola J, Rivero L, Álvarez H, Valdez A, Rodríguez D, Scull R, Payrol J (2006): [Evaluation of antimalarial activity of some plants using in the cuban traditional medicine]. Rev Ciênc Farm Básica Apl 27: 197–205.
- Shioji T, Ueda-Nakamura T, Aparício D, Prado B, Morgado-Díaz JA, de Souza W (2005): Antileishmanial activity of parthenolide, a sesquiterpene lactone isolated from Tanacetum parthenium. Antimicrob Agent Chemother 49: 176–182.
- Singh S, Sivakumar R (2004): Challenges and new discoveries in the treatment of leishmaniasis. J Infect Chemother 10: 307–315.
- Sladowski D, Steer SJ, Clothier RH, Balls M (1993): An improved MTT assay. J Immunol Methods 157: 203–207.
- Sudheesh S, Vijayalakshmi NR (2005): Flavonoids from Punica granatum – Potential antiperoxidative agents. Fitoterapia 76: 181–186.
- Sundar S (2001): Drug resistance in Indian visceral leishmaniasis. Trop Med Int Health 6: 849–854.
- Talakal TS, Dwivedi SK, Sharma SR (1995): In vitro and in vivo therapeutic activity of Parthenium hysterophorus against Trypanosoma evansi. Indian J Exp Biol 33: 894–896.
- Vidyashankar C, Noel GJ (2002): Leishmaniasis. Med J 3: 1–19.
- Wurochekke AU, Nok AJ (2004): In vitro anti trypanosomal activity of some medicinal plants used in the treatment of trypanosomosis in Northern Nigeria. African J Biotech 3: 481–483.
- Zheljazkov VD, Cantrell CL, Tekwani B, Khan SI (2008): Content, composition, and bioactivity of the essential oils of three basil genotypes as a function of harvesting. J Agric Food Chem 56: 380–385.