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Antileishmanial activity of Opuntia ficus-indica fractions
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ARTICLE IN PRESS Biomedicine & Preventive Nutrition xxx (2014) xxx–xxx
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Original article
Antileishmanial activity of Opuntia ficus-indica fractions A. Bargougui a,b,d , P. Champy b , S. Triki d , C. Bories a , P. Le Pape c , P.M. Loiseau a,∗ a Chimiothérapie antiparasitaire, UMR 8076 CNRS BioCIS, Faculté de Pharmacie, University Paris-Sud, rue J. B. Clément, 92296 Châtenay-Malabry cedex, France b Chimie des substances naturelles, UMR 8076 CNRS BioCIS, Faculté de Pharmacie, University Paris-Sud, rue J. B. Clément, 92296 Châtenay-Malabry cedex, France c Département de Parasitologie et Mycologie Médicale, EA 1155 IICiMed, UFR des Sciences Pharmaceutiques et Biologiques Nantes Atlantique Universités, 1, rue Gaston-Veil, 44035 Nantes cedex, France d Laboratoire de Biochimie, Faculté des Sciences, Campus Universitaire, BP 2092, El Manar Tunis, Tunisia
a r t i c l e
i n f o
Article history: Received 27 August 2013 Accepted 16 September 2013 Keywords: Opuntia ficus-indica Antileishmanial activity Trypanocidal activity Bioguided fractionation
a b s t r a c t Cladodes and fruits of Opuntia ficus-indica are used in traditional medicine for the treatment of abscess and skin inflammation. It was therefore interesting to assess whether an antileishmanial activity could be associated to skin healing. This study reports on the antileishmanial activity of Opuntia ficus-indica extracts from cladodes and fruits. Ethyl acetate extract from cladodes only exhibited an activity against Leishmania major with an IC50 value of 53.9 g/mL, but ethyl acetate fruit extract, ethyl acetate cladode extract and methanol cladode extract were active also against Leishmania donovani with IC50 values at 70.3, 70.5 and 45.2 g/mL, respectively. A poor activity of the fractions was monitored against Trypanosoma brucei brucei. Finally, a bioguided fractionation of fruits of Opuntia ficus-indica led to a pre-purified fraction that exhibited an IC50 of 9.3 g/mL against Leishmania donovani intramacrophage amastigotes. The selectivity index defined as CC50 /IC50 was higher than 10. In conclusion, the bioguided fractionation allowed to enhance the antileishmanial activity about ten-fold comparatively to those of the ethyl acetate fruit extract. Such an activity is worth of further investigations to identify the compounds responsible for the antileishmanial effect. © 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction Tropical diseases annually affect millions of people worldwide, being the source of disability and mortality for concerned populations. Among them, leishmaniasis and African trypanosomiasis are widespread in Africa, causing the death of about 60,000 patients for visceral leishmaniasis and about 500,000 new cases each year worldwide [1] and the death of less than 10,000 for African human trypanosomiasis [2]. Their treatments are often toxic and responsible for drug resistance. It is therefore necessary to find new compounds for the treatment of these diseases, giving new opportunities for the control of leishmaniasis and African trypanosomiasis. One way to find new chemical scaffold is to perform a biological screening from plants traditionally used for other health indication. The cladodes and fruits of the cactus Opuntia ficus-indica L. are used in traditional medicine for the treatment of wound healing and abscess, in skin inflammation, eye inflammation, ringworm,
∗ Corresponding author. University Paris-Sud, Faculté de Pharmacie, Groupe Chimiothérapie antiparastaire, 5, rue Jean-Baptiste-Clément, 92290 ChâtenayMalabry, France. Tel.: +33 1 46 83 55 53; fax: +33 1 46 83 55 57. E-mail address: [email protected] (P.M. Loiseau).
as well as for rheumatism, cysts and to detoxify the liver [3]. The presence of cutaneous and visceral leishmaniasis in Tunisia is well documented [4,5] and justifies the search for compounds that could exhibit antileishmanial activity associated to skin healing due to both strains. For this reason, we proposed to investigate the antileishmanial potential of the cladodes and fruits of the cactus Opuntia ficus-indica, cultivar Ain Amara. This preliminary study reports the screening and bioguided fractionation of several extracts of the plant and their antileishmanial activity against L. major and L. mexicana. In addition, the evaluation was extended to L. donovani, responsible for visceral leishmaniasis and Trypanosoma brucei brucei, responsible for African trypanosomiasis in cattle.
2. Materials and methods 2.1. General experimental procedures For column chromatography, Merck Silica gel 60, 70–230 mesh, Reveleris silica cartridge (40 g; 63–200 mesh), were used. Flash chromatography was performed with an Armen Instrument Spot Liquid Chromatography Flash. Analytical TLC were carried out on aluminium plates coated with silica gel 60 F254 (Merck), and
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visualized with UV light, vanillin–H2 SO4 and AlCl3 . Preparative TLC was carried out on glass plate coated with silica gel 60 (Merck). 2.2. Plant material Cladodes of Opuntia ficus-indica L. (Ain Amara cultivar) were harvested at early stage (2–3 weeks-old, devoid of spines) from the FAO collection in Tunisia, in August 2010. Fruits were harvested from the same plants. Epicarps were discarded. Dried voucher specimens (BA Ofi ainamara cl. 2010.08; BA Ofi ainamara fr. 2010.08) were kept at the Faculté des Sciences, El Manar, Tunisia. 2.3. Extraction and purification procedure Fresh cladodes (1 kg) were crushed using a blender. After centrifugation (1 000 g, 1 h), extraction of the pellet (100 g) was performed overnight with MeOH (100 mL), EtOAc (100 mL) or water (100 mL), at room temperature, for 24 h under constant shaking to yield brown-green extracts (MeOH: 9.5 g; EtOAc: 0.3 g; H2 O: 10.0 g). EtOAc extract (2.5 g) was chromatographed on open silica gel column (length 10 cm, diameter 2 cm), using the gradient: EtOAc/C6 H14 1:9, EtOAc/C6 H14 2:8, EtOAc/C6 H14 4:6, EtOAc/C6 H14 5:5, EtOAc/C6 H14 7:3, EtOAc. Then, 30 mL fractions were collected, and grouped according to their TLC profile, yielding 6 fractions named F1 to F6, with the following weights: 452.0; 100.8; 70.5; 57.0; 95.3; 614.0 mg, respectively. Fresh fruit pulp (1 kg) was crushed using a blender and seeds were discarded. After centrifugation (1 000 g, 1 h), extraction of the pellet (182 g) was performed overnight with MeOH (100 mL), EtOAc (100 mL) or water (100 mL), at room temperature, 24 h under constant shaking to yield brown-green extracts (MeOH: 15,0 g; EtOAc: 2.9 g; H2 O: 13.6 g). EtOAc extract (2.93 g) was chromatographed on silica gel (Open column, length 10 cm, diameter 2 cm; Silica gel normal – Silica gel 60,006 – 0.2 mm for column chromatography 70–230 mesh ASTM), using the gradient: EtOAc/C6 H14 1:9, EtOAc/C6 H14 2:8, EtOAc/C6 H14 4:6, EtOAc/C6 H14 5:5, EtOAc/C6 H14 7:3, EtOAc. Then, 30 mL fractions were collected, and grouped according to their TLC profile, yielding 7 fractions named F 1 to F 7, which weights were: 196.0; 304.0; 84.7; 66.5; 79.3; 209.2; 451.4 mg, respectively. Fraction F5 of the fruit EtOAc extract (209.9 mg) underwent automated flash chromatography at 13 bars on a silica Reveleris cartridge, at flow rate 35 mL/min, using the following step gradient: Toluene (5 min), Toluene/CH2 Cl2 7:3 (10 min), CH2 Cl2 (10 min), CH2 Cl2 /MeOH 9.5:0.5 (10 min). Then, 5 mL fractions were collected, and grouped according to their TLC profile, yielding 6 sub-fractions named F 5-1 to F 56 which weights were: 18.8; 13.5; 30.2; 25.6; 96.4; 12.7 mg, respectively. 2.4. In vitro antileishmanial activity on Leishmania major (MHOM/SEN/96/NAN2) and Leishmania mexicana (MHOM/MX/95/NAN1) promastigotes The promastigotes were cultured at 26 ◦ C in Schneider’s insect media (Sigma Chemical Co., St. Louis, MO), supplemented with 15% of fetal calf serum (Sigma), penicillin (100 UI/mL) and streptomycin (100 g/mL). L. major and L. mexicana promastigotes (2.106 parasites/mL) in the late exponential growth phase were inoculated into 96-well plates and exposed to triplicate concentrations of extracts or fractions. The cultures were incubated for 96 h at 26 ◦ C. Each concentration was screened in triplicate. After a 96 h incubation period, the viability of promastigotes was checked using the rezasurin spectrofluorimetric method. Four hours before measurement, 10 L of resazurin solution (700 M) were added. Then fluorescence was measured with the Fluorolite 1000 plate reader
(Dynatech) with an excitation wavelength of 550 nm and an emission wavelength of 590 nm. The fluorescence intensity produced is directly proportional to the number of viable cells. The results are expressed as IC50 , the concentrations inhibiting parasite growth by 50% [6]. Amphotericin B or pentamidine were used as the antileishmanial reference compounds. 2.5. In vitro antileishmanial evaluation against Leishmania donovani (MHOM/ET/67/HU3) 2.5.1. Evaluation against promastigote forms Promastigote forms of L. donovani (MHOM/ET/67/HU3) were grown in M-199 medium supplemented with 40 mM HEPES, 100 M adenosine, 0.5 mg/l hemin, 10% heat-inactivated foetal bovine serum (FBS) and 50 g/mL gentamycin at 26 ◦ C in a dark environment under an atmosphere of 5% CO2 . All the experiments were performed with parasites in their logarithmic phase of growth. Flat-bottomed 96-well microtiter plates maintained at 26 ◦ C in an atmosphere of 95% air/5% CO2 . Two hundred microlitres of culture medium were placed in each well that contains the maximum concentration of the compound to be tested (C1), and 100 L in the following (C2–C7 and controls); 2 L of the stock solution of each fraction dissolved in DMSO was added in C1, and a serial dilution in the well was performed. After 1 h at 27 ◦ C under a 5% CO2 atmosphere, 100 L of the culture medium complemented with 1.75 × 106 promastigotes/mL from a logarithmic phase culture was added to the wells. The final volume in each well was 200 L. Biological tests were performed three times and each tested concentration in triplicate. The viability of parasites was evaluated by using the tetrazolium-dye (MTT) colorimetric method. The MTT cell proliferation assay is a colorimetric assay system, which measures the reduction of a tetrazolium component (MTT) into an insoluble formazan product by the mitochondria of viable cells. After incubation of the cells with the MTT reagent, a detergent solution was added to lyse the cells and solubilize the coloured crystals of formazan. The samples were then read using an ELISA plate reader at a wavelength of 570 nm. The amount of colour produced is directly proportional to the number of viable cells. The results are expressed as the concentration inhibiting parasite growth by 50% (IC50 ) after a 3-day incubation period. Amphotericin B, and pentamidine were used as the antileishmanial reference compounds [7]. 2.5.2. Evaluation against intramacrophage amastigote forms Peritoneal macrophages were harvested from Swiss mice (18–20 g, Janvier, Le Genest Saint Isle, France) three days after an intraperitoneal injection of 1.5 mL of a 3% sodium thioglycolate solution. Mouse macrophages were plated in Labtek® eightchambers slides at 5 × 104 cells per well in 400 L of complete M-199 medium. After 24 h at 37 ◦ C in an atmosphere of 5% CO2 in air, the macrophages were infected with promastigotes of L. donovani at the stationary growth phase from 7-day-old cultures at a ratio of 20 parasites per macrophage. After washing to eliminate excess parasites, the cells were incubated for 24 h in culture medium alone. Then, the medium was discarded and the cells were incubated with the different fractions at various concentrations. After 48 h of treatment, the medium and the chambers were removed and the slides were fixed with MeOH and Giemsa-stained. The number of amastigotes in 100 macrophages/coverslip was determined microscopically and the IC50 (M) was determined by linear regression comparatively to untreated controls [7]. 2.6. In vitro trypanocidal evaluation against Trypanosoma brucei brucei CMP fast strain The method used was previously described by Loiseau et al. [8]. T. b. brucei CMP is a trypanosome fast strain leading to the death of
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Table 1 In vitro antileishmanial and trypanocidal activities and cytotoxicity on MRC5 and HeLa cells of extract of Opuntia ficus-indica. Extracts
L.major promastigotes IC50 ± SD (g/mL)
L. mexicana promastigotes IC50 ± SD (g/mL)
L.donovani promastigotes IC50 ± SD (g/mL)
T. brucei trypomastigotes MEC (g/mL)
MRC5 CC50 ± SD (g/mL)
Hela C50 ± SD (g/mL)
Amphotericin B
> 500 > 500 53.9 ± 8.3 > 500 0.04 ± 0.01 M
> 500 > 500 109.8 ± 13.1 243.9 ± 23.3 /
> 125 > 125 125 125 /
/ / 164.6 ± 15.5 223.7 ± 56.4 /
/ / 133.9 ± 88.6 79.2 ± 25.5 /
Pentamidine
/
6.3 ± 0.4 M
45.2 ± 3.8 > 100 70.5 ± 7.4 70.3 ± 6.9 0.078 ± 0.006 M 2.8 ± 0.3 M
3.1
/
/
Methanol Ethyl acetate
Cladodes Fruit Cladodes Fruit
IC50 : Inhibitory concentration 50% ± SD; CC50 : Cytotoxic concentration 50% ± SD. MEC: Minimum effective concentration, Water extracts were inactive: IC50 > 500 g/mL.
mice three days after infection. Briefly, blood from infected mice (18–20 g, Janvier, Le Genest Saint Isle, France) was collected aseptically from the retro-orbital sinus and the bloodstream forms of T. brucei brucei were purified from a centrifugation (900 g, 4 ◦ C). Trypanosomes were maintained in vitro for 24 h in the dark at 37 ◦ C in a 5% CO2 atmosphere, in minimum essential medium (Gibco BRL) including 25 mM HEPES and Earle’s salts and supplemented with 2 mM glutamine, 1 g of additional glucose per liter, 10 mL of minimum essential medium non-essential amino acids (100×; Gibco BRL) per l, 0.2 mM 2-mercaptoethanol, 2 mM sodium pyruvate, 0.1 mM hypoxanthine, 0.016 mM thymidine, 15% heat-inactivated horse serum (Gibco BRL), and 50 g of gentamycin per mL. The trypanocidal evaluation of the fractions was carried out in 96-well tissue culture plates in a final volume of 200 L containing 2 × 105 trypomastigotes previously purified by centrifugation (900 g, 4 ◦ C) from the blood of an infected Swiss mouse, and the compounds to be tested. Pentamidine was used as the reference compound. The minimum effective concentration (MEC) was defined as the minimum concentration at which no viable parasite was observed microscopically. This value was confirmed by injecting intraperitoneally the culture from the well corresponding to the MEC into a Swiss mouse by intraperitoneal route to confirm that the nonmotile parasites were really killed and not able to divide into the mice. 2.7. Cytotoxicity HeLa and MRC5 cells were subcultured every 4 days in RPMI 1640 medium supplemented with 10% FBS (Sigma–Aldrich). The cells were harvested after a 5-min incubation with trypsine solution (Sigma–Aldrich). HeLa and MRC5 cells were seeded in a 96-well microplate, 100 L of a 105 /mL suspension in each well. After a 24 h incubation time at 37 ◦ C and 5% CO2 , 100 L of the extract concentrations were added in duplicate. After 96 h, 10 L of resazurin solution (700 M) were added. After a 4 h incubation at 37 ◦ C, 5% CO2 , fluorescence was measured at 590 nm with an excitation at 550 nm. 3. Results and discussion The cladodes and fruits of the cactus Opuntia ficus-indica L. are used in traditional medicine to treat skin inflammation and abscess [3]. It was therefore interesting to search for an antileishmanial activity associated to skin healing. Water, methanol and ethyl acetate extracts of cladodes and fruit pulp of Opuntia ficus-indica were evaluated against the promastigote forms of L. major, responsible for cutaneous leishmaniasis and the study was extended to L. mexicana and L. donovani and the trypomastigote forms of T. brucei brucei in order to select the more active extracts for further fractionation. Cell viability was determined on L. major and L. mexicana by using a resazurin spectrofluorimetric method in
Nantes (France) whereas MTT was used for L. donovani in ChatenayMalabry (France). Both the methods are worthwhile and equivalent for IC50 determinations. The antiparasitic results obtained with the extracts are gathered in Table 1. The most active extract against the three species of Leishmania and against T. b. brucei was ethyl acetate extract from cladodes with IC50 values in a range from 53.9 to 109.8 g/mL on the three Leishmania species (L. major, L. mexicana and L. donovani) and a MEC value at 125 g/mL on T. brucei. The ethyl acetate extract from fruit pulp was active against L. donovani (IC50 value at 70.3 g/mL) and slightly active against L. mexicana (IC50 value at 243.9 g/mL). In addition, this extract was also active against T. b. brucei (MEC value at 125 g/mL). Water and methanol extracts were inactive against Leishmania species and T. b. brucei, except the methanol extract of cladodes that exhibited an IC50 at 45.2 ± 3.8 g/mL on L. donovani promastigotes. Considering the level of activities against the three Leishmania strains, we prioritized the ethyl acetate cladodes and fruit extracts for further purifications. We also checked the cytotoxicity of both the ethyl acetate extracts on MRC5 and HeLa cell lines (Table 1). The results showed that both the extracts contained some toxic compounds for the cells (MIC in a range from 79.2 to 223.7 g/mL). We continued the fractionation on the less toxic extract, the fruit pulp ethyl acetate extract. This was carried out by liquid chromatography at atmospheric pressure in order to identify the most active antileishmanial fractions. Fraction F 5 from fruit was the most active against L. donovani with an IC50 value of 18.3 g/mL (Table 2). This fraction was slightly active against T. b. brucei (MEC value at 125 g/mL). Fraction F 5 was therefore considered for sub-fractionation using silica gel Table 2 Antileishmanial and trypanocidal activities of fractions obtained from Opuntia ficusindica. Fractions
Cladodes F1 F2 F3 F4 F5 F6 Fruit F 1 F 2 F 3 F 4 F 5 F 6 F 7 Amphotericin B Pentamidine
Leishmania donovani promastigotes IC50 (g/mL) ± SD
Trypanosoma brucei brucei trypomastigotes MEC (g/mL)
> 100 > 100 80.6 ± 9.3 > 100 > 100 50.4 ± 5.1
> 125 > 125 > 125 > 125 > 125 > 125
> 100 > 100 37.1 ± 2.7 30.4 ± 2.9 18.3 ± 2.1 > 100 75.1 ± 8.2 0.078 ± 0.008 M 2.8 ± 0.3 M
> 125 > 125 62.5 125 125 125 > 125 / 3.1 M
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Table 3 Antileishmanial and trypanocidal activities of sub-fractions from F 5 obtained from fruits of Opuntia ficus-indica. Sub-fractions
F 5-1 F 5-2 F 5-3 F 5-4 F 5-5 F 5-6 Amphotericin B Pentamidine
L. donovani promastigotes IC50 (g/mL) +SD
L. donovani intramacrophage amastigotes IC50 (g/mL) +SD
T. brucei brucei trypomastigotes MEC (g/mL)
> 100 75.3 ± 8.4 > 100 25.3 ± 1.9 6.2 ± 0.7 > 100 0.078 ± 0.007 M 2.8 ± 0.3 M
> 100 > 100 > 100 43.1 ± 2.9 9.3 ± 1.2 > 100 0.094 ± 0.007 13.4 ± 1.1
125 125 31.3 > 125 62.5 62.5 / 3.1 M
In conclusion, this plant that was traditionally used in the treatment of abscess and skin inflammation exhibits antileishmanial activity. Such combined effects could have a future in the treatment of disseminated leishmaniasis. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgements Franc¸oise Huteau and Fabrice Pagniez are acknowledged for their help in cell culture and in vitro antiparasitic evaluation. References
chromatography. Among the six sub-fractions obtained from this separation, the most antileishmanial sub-fraction was sub-fraction F 5-5 with an IC50 value of 6.2 g/mL against L. donovani and a MEC value of 62.5 g/mL against T. brucei. Sub-fraction F 5-3 exhibited a MEC value at 31.3 g/mL against T. b. brucei. Sub-fraction F 5-5 was then evaluated against intramacrophage amastigotes of L. donovani and the IC50 value was 9.3 g/mL. Such an activity was slightly better than those of pentamidine, used as the reference compound. No toxicity was observed on murine peritoneal macrophages at 100 g/mL. Thus, the selectivity index defined as CC50 /IC50 is higher than 10. In addition, the bioguided fractionation allowed to enhance the antileishmanial activity about ten-fold comparatively to the ethyl acetate fruit extract. Some studies about the chemical composition of the plant highlighted the presence of flavonoids [9,10]. As flavonoids are known to exhibit antileishmanial activity, the isolation of the antileishmanial agents are required to verify the importance of flavonoids in the antileishmanial properties [11,12] (Table 3). 4. Conclusion Extracts of cladodes and fruits of Opuntia ficus-indica exhibited a significant antileishmanial activity whereas a poor trypanocidal activity. The bioguided fractionation of fruits of Opuntia ficusindica led to pre-purified fractions that were very active against L. donovani intramacrophage amastigotes. This is the first time that such an activity is described for this plant. Further studies will be focused on the isolation and identification of the active principle from sub-fraction F 5-5. In addition, ethyl acetate cladode extract that was active against L. major is worth of further fractionation.
[1] Pham TTH, Loiseau PM, Barratt G. Strategies for the design of orally bioavailable antileishmanial treatments. Int J Pharm 2013, http://dx.doi.org/10.1016/j.ijpharm.2013.07.035. [2] Simarro PP, Diarra A, Postigo JAR, Franco JR, Jannin JG. The human African trypanosomiasis control and surveillance program of the World Heath Organization 2000-2009: the way forward. PLOS Negl Trop Dis 2011;5:e1007. [3] Le Floc’h E. Contribution à une étude éthnobotanique de la flore tunisienne. Imprimerie Officielle Repub Tunisienne 1983;2:161–2. [4] Aoun K, Ben Abda I, Bousslimi N, Bettaieb J, Siala E, Ben Abdallah R, et al. Bouratbine. Comparative characterization of skin lesions observed in the three endemic varieties of cutaneous leishmaniasis in Tunisia. Ann Dermatol Venereol 2012;139:452–8. [5] Ben Salah A, Ben Ismail R, Amri F, Schlif S, Ben Rzig F, Kharrat H, et al. Investigation of the spread of human visceral leishmaniasis in central Tunisia. Trans Roy Soc Trop Med Hyg 2000;94:382–6. [6] Le Pape P, Pagniez F, Abdala-Valencia H. A new automatized fluorometric assay for anti-Leishmania drug screening. Acta Parasitologica 2002;47:79–81. [7] Chollet C, Crousse B, Bories C, Bonnet-Delpon D, Loiseau PM. In vitro antileishmanial evaluation of fluoro-artemisinin derivatives against Leishmania donovani. Biomed Pharmacother 2008;62:462–5. [8] Loiseau PM, Lubert P, Wolf JG. Contribution of dithiols ligands to in vitro and in vivo trypanocidal activities of dithiaarsanes and investigation of ligand exchange in an aqueous solution. Antimicrob Agents Chemother 2000;44:2954–61. [9] Dok-Go H, Lee KH, Kim HJ, Lee EH, Lee J, Song YS, et al. Neuroprotective effects of antioxidative flavonoids, quercetin, (+)-dihydroquercetin and quercetin 3-methyl ether, isolated from Opuntia ficus-indica var. saboten. Brain Res 2003;965:130–6. [10] Diaz-Medina EM, Rodriguez-Rodríguez EM, Diaz-Romero C. Chemical characterization of Opuntia dillenii and Opuntia ficus-indica fruits. Food Chem 2007;103:38–45. [11] Grecco SS, Reimão JQ, Tempone AG, Sartorelli P, Romoff P, Ferreira MJ, et al. Isolation of an antileishmanial and antitrypanosomal flavanone from the leaves of Baccharis retusa DC (Asteraceae). Parasitol Res 2010;106: 1245–8. [12] Sartorelli P, Carvalho CS, Reimão JQ, Ferreira MJ, Tempone AG. Antiparasitic activity of biochanin A, an isolated isoflavone from fruits of Cassia fistula (Leguminosae). Parasitol Res 2009;104:311–4.
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Original article
Antileishmanial activity of Opuntia ficus-indica fractions A. Bargougui a,b,d , P. Champy b , S. Triki d , C. Bories a , P. Le Pape c , P.M. Loiseau a,∗ a Chimiothérapie antiparasitaire, UMR 8076 CNRS BioCIS, Faculté de Pharmacie, University Paris-Sud, rue J. B. Clément, 92296 Châtenay-Malabry cedex, France b Chimie des substances naturelles, UMR 8076 CNRS BioCIS, Faculté de Pharmacie, University Paris-Sud, rue J. B. Clément, 92296 Châtenay-Malabry cedex, France c Département de Parasitologie et Mycologie Médicale, EA 1155 IICiMed, UFR des Sciences Pharmaceutiques et Biologiques Nantes Atlantique Universités, 1, rue Gaston-Veil, 44035 Nantes cedex, France d Laboratoire de Biochimie, Faculté des Sciences, Campus Universitaire, BP 2092, El Manar Tunis, Tunisia
a r t i c l e
i n f o
Article history: Received 27 August 2013 Accepted 16 September 2013 Keywords: Opuntia ficus-indica Antileishmanial activity Trypanocidal activity Bioguided fractionation
a b s t r a c t Cladodes and fruits of Opuntia ficus-indica are used in traditional medicine for the treatment of abscess and skin inflammation. It was therefore interesting to assess whether an antileishmanial activity could be associated to skin healing. This study reports on the antileishmanial activity of Opuntia ficus-indica extracts from cladodes and fruits. Ethyl acetate extract from cladodes only exhibited an activity against Leishmania major with an IC50 value of 53.9 g/mL, but ethyl acetate fruit extract, ethyl acetate cladode extract and methanol cladode extract were active also against Leishmania donovani with IC50 values at 70.3, 70.5 and 45.2 g/mL, respectively. A poor activity of the fractions was monitored against Trypanosoma brucei brucei. Finally, a bioguided fractionation of fruits of Opuntia ficus-indica led to a pre-purified fraction that exhibited an IC50 of 9.3 g/mL against Leishmania donovani intramacrophage amastigotes. The selectivity index defined as CC50 /IC50 was higher than 10. In conclusion, the bioguided fractionation allowed to enhance the antileishmanial activity about ten-fold comparatively to those of the ethyl acetate fruit extract. Such an activity is worth of further investigations to identify the compounds responsible for the antileishmanial effect. © 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction Tropical diseases annually affect millions of people worldwide, being the source of disability and mortality for concerned populations. Among them, leishmaniasis and African trypanosomiasis are widespread in Africa, causing the death of about 60,000 patients for visceral leishmaniasis and about 500,000 new cases each year worldwide [1] and the death of less than 10,000 for African human trypanosomiasis [2]. Their treatments are often toxic and responsible for drug resistance. It is therefore necessary to find new compounds for the treatment of these diseases, giving new opportunities for the control of leishmaniasis and African trypanosomiasis. One way to find new chemical scaffold is to perform a biological screening from plants traditionally used for other health indication. The cladodes and fruits of the cactus Opuntia ficus-indica L. are used in traditional medicine for the treatment of wound healing and abscess, in skin inflammation, eye inflammation, ringworm,
∗ Corresponding author. University Paris-Sud, Faculté de Pharmacie, Groupe Chimiothérapie antiparastaire, 5, rue Jean-Baptiste-Clément, 92290 ChâtenayMalabry, France. Tel.: +33 1 46 83 55 53; fax: +33 1 46 83 55 57. E-mail address: [email protected] (P.M. Loiseau).
as well as for rheumatism, cysts and to detoxify the liver [3]. The presence of cutaneous and visceral leishmaniasis in Tunisia is well documented [4,5] and justifies the search for compounds that could exhibit antileishmanial activity associated to skin healing due to both strains. For this reason, we proposed to investigate the antileishmanial potential of the cladodes and fruits of the cactus Opuntia ficus-indica, cultivar Ain Amara. This preliminary study reports the screening and bioguided fractionation of several extracts of the plant and their antileishmanial activity against L. major and L. mexicana. In addition, the evaluation was extended to L. donovani, responsible for visceral leishmaniasis and Trypanosoma brucei brucei, responsible for African trypanosomiasis in cattle.
2. Materials and methods 2.1. General experimental procedures For column chromatography, Merck Silica gel 60, 70–230 mesh, Reveleris silica cartridge (40 g; 63–200 mesh), were used. Flash chromatography was performed with an Armen Instrument Spot Liquid Chromatography Flash. Analytical TLC were carried out on aluminium plates coated with silica gel 60 F254 (Merck), and
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visualized with UV light, vanillin–H2 SO4 and AlCl3 . Preparative TLC was carried out on glass plate coated with silica gel 60 (Merck). 2.2. Plant material Cladodes of Opuntia ficus-indica L. (Ain Amara cultivar) were harvested at early stage (2–3 weeks-old, devoid of spines) from the FAO collection in Tunisia, in August 2010. Fruits were harvested from the same plants. Epicarps were discarded. Dried voucher specimens (BA Ofi ainamara cl. 2010.08; BA Ofi ainamara fr. 2010.08) were kept at the Faculté des Sciences, El Manar, Tunisia. 2.3. Extraction and purification procedure Fresh cladodes (1 kg) were crushed using a blender. After centrifugation (1 000 g, 1 h), extraction of the pellet (100 g) was performed overnight with MeOH (100 mL), EtOAc (100 mL) or water (100 mL), at room temperature, for 24 h under constant shaking to yield brown-green extracts (MeOH: 9.5 g; EtOAc: 0.3 g; H2 O: 10.0 g). EtOAc extract (2.5 g) was chromatographed on open silica gel column (length 10 cm, diameter 2 cm), using the gradient: EtOAc/C6 H14 1:9, EtOAc/C6 H14 2:8, EtOAc/C6 H14 4:6, EtOAc/C6 H14 5:5, EtOAc/C6 H14 7:3, EtOAc. Then, 30 mL fractions were collected, and grouped according to their TLC profile, yielding 6 fractions named F1 to F6, with the following weights: 452.0; 100.8; 70.5; 57.0; 95.3; 614.0 mg, respectively. Fresh fruit pulp (1 kg) was crushed using a blender and seeds were discarded. After centrifugation (1 000 g, 1 h), extraction of the pellet (182 g) was performed overnight with MeOH (100 mL), EtOAc (100 mL) or water (100 mL), at room temperature, 24 h under constant shaking to yield brown-green extracts (MeOH: 15,0 g; EtOAc: 2.9 g; H2 O: 13.6 g). EtOAc extract (2.93 g) was chromatographed on silica gel (Open column, length 10 cm, diameter 2 cm; Silica gel normal – Silica gel 60,006 – 0.2 mm for column chromatography 70–230 mesh ASTM), using the gradient: EtOAc/C6 H14 1:9, EtOAc/C6 H14 2:8, EtOAc/C6 H14 4:6, EtOAc/C6 H14 5:5, EtOAc/C6 H14 7:3, EtOAc. Then, 30 mL fractions were collected, and grouped according to their TLC profile, yielding 7 fractions named F 1 to F 7, which weights were: 196.0; 304.0; 84.7; 66.5; 79.3; 209.2; 451.4 mg, respectively. Fraction F5 of the fruit EtOAc extract (209.9 mg) underwent automated flash chromatography at 13 bars on a silica Reveleris cartridge, at flow rate 35 mL/min, using the following step gradient: Toluene (5 min), Toluene/CH2 Cl2 7:3 (10 min), CH2 Cl2 (10 min), CH2 Cl2 /MeOH 9.5:0.5 (10 min). Then, 5 mL fractions were collected, and grouped according to their TLC profile, yielding 6 sub-fractions named F 5-1 to F 56 which weights were: 18.8; 13.5; 30.2; 25.6; 96.4; 12.7 mg, respectively. 2.4. In vitro antileishmanial activity on Leishmania major (MHOM/SEN/96/NAN2) and Leishmania mexicana (MHOM/MX/95/NAN1) promastigotes The promastigotes were cultured at 26 ◦ C in Schneider’s insect media (Sigma Chemical Co., St. Louis, MO), supplemented with 15% of fetal calf serum (Sigma), penicillin (100 UI/mL) and streptomycin (100 g/mL). L. major and L. mexicana promastigotes (2.106 parasites/mL) in the late exponential growth phase were inoculated into 96-well plates and exposed to triplicate concentrations of extracts or fractions. The cultures were incubated for 96 h at 26 ◦ C. Each concentration was screened in triplicate. After a 96 h incubation period, the viability of promastigotes was checked using the rezasurin spectrofluorimetric method. Four hours before measurement, 10 L of resazurin solution (700 M) were added. Then fluorescence was measured with the Fluorolite 1000 plate reader
(Dynatech) with an excitation wavelength of 550 nm and an emission wavelength of 590 nm. The fluorescence intensity produced is directly proportional to the number of viable cells. The results are expressed as IC50 , the concentrations inhibiting parasite growth by 50% [6]. Amphotericin B or pentamidine were used as the antileishmanial reference compounds. 2.5. In vitro antileishmanial evaluation against Leishmania donovani (MHOM/ET/67/HU3) 2.5.1. Evaluation against promastigote forms Promastigote forms of L. donovani (MHOM/ET/67/HU3) were grown in M-199 medium supplemented with 40 mM HEPES, 100 M adenosine, 0.5 mg/l hemin, 10% heat-inactivated foetal bovine serum (FBS) and 50 g/mL gentamycin at 26 ◦ C in a dark environment under an atmosphere of 5% CO2 . All the experiments were performed with parasites in their logarithmic phase of growth. Flat-bottomed 96-well microtiter plates maintained at 26 ◦ C in an atmosphere of 95% air/5% CO2 . Two hundred microlitres of culture medium were placed in each well that contains the maximum concentration of the compound to be tested (C1), and 100 L in the following (C2–C7 and controls); 2 L of the stock solution of each fraction dissolved in DMSO was added in C1, and a serial dilution in the well was performed. After 1 h at 27 ◦ C under a 5% CO2 atmosphere, 100 L of the culture medium complemented with 1.75 × 106 promastigotes/mL from a logarithmic phase culture was added to the wells. The final volume in each well was 200 L. Biological tests were performed three times and each tested concentration in triplicate. The viability of parasites was evaluated by using the tetrazolium-dye (MTT) colorimetric method. The MTT cell proliferation assay is a colorimetric assay system, which measures the reduction of a tetrazolium component (MTT) into an insoluble formazan product by the mitochondria of viable cells. After incubation of the cells with the MTT reagent, a detergent solution was added to lyse the cells and solubilize the coloured crystals of formazan. The samples were then read using an ELISA plate reader at a wavelength of 570 nm. The amount of colour produced is directly proportional to the number of viable cells. The results are expressed as the concentration inhibiting parasite growth by 50% (IC50 ) after a 3-day incubation period. Amphotericin B, and pentamidine were used as the antileishmanial reference compounds [7]. 2.5.2. Evaluation against intramacrophage amastigote forms Peritoneal macrophages were harvested from Swiss mice (18–20 g, Janvier, Le Genest Saint Isle, France) three days after an intraperitoneal injection of 1.5 mL of a 3% sodium thioglycolate solution. Mouse macrophages were plated in Labtek® eightchambers slides at 5 × 104 cells per well in 400 L of complete M-199 medium. After 24 h at 37 ◦ C in an atmosphere of 5% CO2 in air, the macrophages were infected with promastigotes of L. donovani at the stationary growth phase from 7-day-old cultures at a ratio of 20 parasites per macrophage. After washing to eliminate excess parasites, the cells were incubated for 24 h in culture medium alone. Then, the medium was discarded and the cells were incubated with the different fractions at various concentrations. After 48 h of treatment, the medium and the chambers were removed and the slides were fixed with MeOH and Giemsa-stained. The number of amastigotes in 100 macrophages/coverslip was determined microscopically and the IC50 (M) was determined by linear regression comparatively to untreated controls [7]. 2.6. In vitro trypanocidal evaluation against Trypanosoma brucei brucei CMP fast strain The method used was previously described by Loiseau et al. [8]. T. b. brucei CMP is a trypanosome fast strain leading to the death of
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Table 1 In vitro antileishmanial and trypanocidal activities and cytotoxicity on MRC5 and HeLa cells of extract of Opuntia ficus-indica. Extracts
L.major promastigotes IC50 ± SD (g/mL)
L. mexicana promastigotes IC50 ± SD (g/mL)
L.donovani promastigotes IC50 ± SD (g/mL)
T. brucei trypomastigotes MEC (g/mL)
MRC5 CC50 ± SD (g/mL)
Hela C50 ± SD (g/mL)
Amphotericin B
> 500 > 500 53.9 ± 8.3 > 500 0.04 ± 0.01 M
> 500 > 500 109.8 ± 13.1 243.9 ± 23.3 /
> 125 > 125 125 125 /
/ / 164.6 ± 15.5 223.7 ± 56.4 /
/ / 133.9 ± 88.6 79.2 ± 25.5 /
Pentamidine
/
6.3 ± 0.4 M
45.2 ± 3.8 > 100 70.5 ± 7.4 70.3 ± 6.9 0.078 ± 0.006 M 2.8 ± 0.3 M
3.1
/
/
Methanol Ethyl acetate
Cladodes Fruit Cladodes Fruit
IC50 : Inhibitory concentration 50% ± SD; CC50 : Cytotoxic concentration 50% ± SD. MEC: Minimum effective concentration, Water extracts were inactive: IC50 > 500 g/mL.
mice three days after infection. Briefly, blood from infected mice (18–20 g, Janvier, Le Genest Saint Isle, France) was collected aseptically from the retro-orbital sinus and the bloodstream forms of T. brucei brucei were purified from a centrifugation (900 g, 4 ◦ C). Trypanosomes were maintained in vitro for 24 h in the dark at 37 ◦ C in a 5% CO2 atmosphere, in minimum essential medium (Gibco BRL) including 25 mM HEPES and Earle’s salts and supplemented with 2 mM glutamine, 1 g of additional glucose per liter, 10 mL of minimum essential medium non-essential amino acids (100×; Gibco BRL) per l, 0.2 mM 2-mercaptoethanol, 2 mM sodium pyruvate, 0.1 mM hypoxanthine, 0.016 mM thymidine, 15% heat-inactivated horse serum (Gibco BRL), and 50 g of gentamycin per mL. The trypanocidal evaluation of the fractions was carried out in 96-well tissue culture plates in a final volume of 200 L containing 2 × 105 trypomastigotes previously purified by centrifugation (900 g, 4 ◦ C) from the blood of an infected Swiss mouse, and the compounds to be tested. Pentamidine was used as the reference compound. The minimum effective concentration (MEC) was defined as the minimum concentration at which no viable parasite was observed microscopically. This value was confirmed by injecting intraperitoneally the culture from the well corresponding to the MEC into a Swiss mouse by intraperitoneal route to confirm that the nonmotile parasites were really killed and not able to divide into the mice. 2.7. Cytotoxicity HeLa and MRC5 cells were subcultured every 4 days in RPMI 1640 medium supplemented with 10% FBS (Sigma–Aldrich). The cells were harvested after a 5-min incubation with trypsine solution (Sigma–Aldrich). HeLa and MRC5 cells were seeded in a 96-well microplate, 100 L of a 105 /mL suspension in each well. After a 24 h incubation time at 37 ◦ C and 5% CO2 , 100 L of the extract concentrations were added in duplicate. After 96 h, 10 L of resazurin solution (700 M) were added. After a 4 h incubation at 37 ◦ C, 5% CO2 , fluorescence was measured at 590 nm with an excitation at 550 nm. 3. Results and discussion The cladodes and fruits of the cactus Opuntia ficus-indica L. are used in traditional medicine to treat skin inflammation and abscess [3]. It was therefore interesting to search for an antileishmanial activity associated to skin healing. Water, methanol and ethyl acetate extracts of cladodes and fruit pulp of Opuntia ficus-indica were evaluated against the promastigote forms of L. major, responsible for cutaneous leishmaniasis and the study was extended to L. mexicana and L. donovani and the trypomastigote forms of T. brucei brucei in order to select the more active extracts for further fractionation. Cell viability was determined on L. major and L. mexicana by using a resazurin spectrofluorimetric method in
Nantes (France) whereas MTT was used for L. donovani in ChatenayMalabry (France). Both the methods are worthwhile and equivalent for IC50 determinations. The antiparasitic results obtained with the extracts are gathered in Table 1. The most active extract against the three species of Leishmania and against T. b. brucei was ethyl acetate extract from cladodes with IC50 values in a range from 53.9 to 109.8 g/mL on the three Leishmania species (L. major, L. mexicana and L. donovani) and a MEC value at 125 g/mL on T. brucei. The ethyl acetate extract from fruit pulp was active against L. donovani (IC50 value at 70.3 g/mL) and slightly active against L. mexicana (IC50 value at 243.9 g/mL). In addition, this extract was also active against T. b. brucei (MEC value at 125 g/mL). Water and methanol extracts were inactive against Leishmania species and T. b. brucei, except the methanol extract of cladodes that exhibited an IC50 at 45.2 ± 3.8 g/mL on L. donovani promastigotes. Considering the level of activities against the three Leishmania strains, we prioritized the ethyl acetate cladodes and fruit extracts for further purifications. We also checked the cytotoxicity of both the ethyl acetate extracts on MRC5 and HeLa cell lines (Table 1). The results showed that both the extracts contained some toxic compounds for the cells (MIC in a range from 79.2 to 223.7 g/mL). We continued the fractionation on the less toxic extract, the fruit pulp ethyl acetate extract. This was carried out by liquid chromatography at atmospheric pressure in order to identify the most active antileishmanial fractions. Fraction F 5 from fruit was the most active against L. donovani with an IC50 value of 18.3 g/mL (Table 2). This fraction was slightly active against T. b. brucei (MEC value at 125 g/mL). Fraction F 5 was therefore considered for sub-fractionation using silica gel Table 2 Antileishmanial and trypanocidal activities of fractions obtained from Opuntia ficusindica. Fractions
Cladodes F1 F2 F3 F4 F5 F6 Fruit F 1 F 2 F 3 F 4 F 5 F 6 F 7 Amphotericin B Pentamidine
Leishmania donovani promastigotes IC50 (g/mL) ± SD
Trypanosoma brucei brucei trypomastigotes MEC (g/mL)
> 100 > 100 80.6 ± 9.3 > 100 > 100 50.4 ± 5.1
> 125 > 125 > 125 > 125 > 125 > 125
> 100 > 100 37.1 ± 2.7 30.4 ± 2.9 18.3 ± 2.1 > 100 75.1 ± 8.2 0.078 ± 0.008 M 2.8 ± 0.3 M
> 125 > 125 62.5 125 125 125 > 125 / 3.1 M
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Table 3 Antileishmanial and trypanocidal activities of sub-fractions from F 5 obtained from fruits of Opuntia ficus-indica. Sub-fractions
F 5-1 F 5-2 F 5-3 F 5-4 F 5-5 F 5-6 Amphotericin B Pentamidine
L. donovani promastigotes IC50 (g/mL) +SD
L. donovani intramacrophage amastigotes IC50 (g/mL) +SD
T. brucei brucei trypomastigotes MEC (g/mL)
> 100 75.3 ± 8.4 > 100 25.3 ± 1.9 6.2 ± 0.7 > 100 0.078 ± 0.007 M 2.8 ± 0.3 M
> 100 > 100 > 100 43.1 ± 2.9 9.3 ± 1.2 > 100 0.094 ± 0.007 13.4 ± 1.1
125 125 31.3 > 125 62.5 62.5 / 3.1 M
In conclusion, this plant that was traditionally used in the treatment of abscess and skin inflammation exhibits antileishmanial activity. Such combined effects could have a future in the treatment of disseminated leishmaniasis. Disclosure of interest The authors declare that they have no conflicts of interest concerning this article. Acknowledgements Franc¸oise Huteau and Fabrice Pagniez are acknowledged for their help in cell culture and in vitro antiparasitic evaluation. References
chromatography. Among the six sub-fractions obtained from this separation, the most antileishmanial sub-fraction was sub-fraction F 5-5 with an IC50 value of 6.2 g/mL against L. donovani and a MEC value of 62.5 g/mL against T. brucei. Sub-fraction F 5-3 exhibited a MEC value at 31.3 g/mL against T. b. brucei. Sub-fraction F 5-5 was then evaluated against intramacrophage amastigotes of L. donovani and the IC50 value was 9.3 g/mL. Such an activity was slightly better than those of pentamidine, used as the reference compound. No toxicity was observed on murine peritoneal macrophages at 100 g/mL. Thus, the selectivity index defined as CC50 /IC50 is higher than 10. In addition, the bioguided fractionation allowed to enhance the antileishmanial activity about ten-fold comparatively to the ethyl acetate fruit extract. Some studies about the chemical composition of the plant highlighted the presence of flavonoids [9,10]. As flavonoids are known to exhibit antileishmanial activity, the isolation of the antileishmanial agents are required to verify the importance of flavonoids in the antileishmanial properties [11,12] (Table 3). 4. Conclusion Extracts of cladodes and fruits of Opuntia ficus-indica exhibited a significant antileishmanial activity whereas a poor trypanocidal activity. The bioguided fractionation of fruits of Opuntia ficusindica led to pre-purified fractions that were very active against L. donovani intramacrophage amastigotes. This is the first time that such an activity is described for this plant. Further studies will be focused on the isolation and identification of the active principle from sub-fraction F 5-5. In addition, ethyl acetate cladode extract that was active against L. major is worth of further fractionation.
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