Experimental Parasitology 141 (2014) 106–111
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Activity of olive leaf extracts against the promastigote stage of Leishmania species and their correlation with the antioxidant activity Ines Sifaoui a,⇑, Atteneri López-Arencibia b,1, Carmen Ma Martín-Navarro b,c,1, Nadia Chammem d,2, María Reyes-Batlle b,1, Mondher Mejri a,3, Jacob Lorenzo-Morales b,1, Manef Abderabba a,3, José E. Piñero b,1 a
Laboratoire Matériaux-Molécules et Applications, IPEST, University of Carthage, B.P. 51, 2070 La Marsa, Tunisia University Institute of Tropical Diseases and Public Health, University of La Laguna, Avda Francisco Sanchez s/n, Campus de Anchieta, 38271 La Laguna, Tenerife, Canary Islands, Spain Centre for Integrative Physiology, University of Edinburgh, Scotland, UK d Laboratoire d’Ecologie et de Technologie Microbienne, INSAT, University of Carthage, Centre Urbain Nord, B.P. 676, 1080 Tunis Cedex, Tunisia b c
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Olive leaf extract present an
interesting leishmanicidal activity. The antiparasitic activity was
significantly affected by the olive cultivar and the tested Leishmania strains. The Limouni variety presented the highest content on phenolic compounds and the stronger leishmanicidal activity. L. amazonensis was the most sensitive strain to the assayed olive leaf extract.
a r t i c l e
i n f o
Article history: Received 17 April 2013 Received in revised form 19 February 2014 Accepted 4 March 2014 Available online 21 March 2014 Keywords: Olive leaf extract Leishmanicidal activity Antioxidant activity
a b s t r a c t Leishmaniasis is one of the neglected tropical diseases in terms of drug discovery and development. Furthermore, the chemotherapy used to treat this disease has been proved to be highly toxic and to present resistance issues. As consequent, the need for novel leishmanicidal molecules has notably increased in the recent years. In the present work an attempt was made to evaluate the antioxidant and leishmanicidal activities besides presence of compounds in leaf extracts of 5 different Tunisian olive tree varieties, used as traditional medicine in this country. The concentration of extracts needed to inhibit 50% of the parasitic growth (IC50) was estimated using different Leishmania strains. All tested extracts showed an inhibitory effect on the parasite growth with IC50 ranging from 2.130 ± 0.023 to 71.570 ± 4.324 lg/ml, respectively for the methanolic extracts of Limouni and Zarrazi against Leishmania donovani. In fact, this activity was significantly affected by the olive cultivar and the tested Leishmania strain. Furthermore, the activities against both Leishmania tropica and major species were correlated to the total phenolic compounds. These results could suggest that olive leaf extract could carry potential new compounds for the development of novel drugs against Leishmaniasis. Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction ⇑ Corresponding author. Fax: +216 71 746 551. 1 2 3
E-mail address: [email protected] (I. Sifaoui). Fax: +34 922318514. Fax: +216 71704329. Fax: +216 71746551.
http://dx.doi.org/10.1016/j.exppara.2014.03.002 0014-4894/Ó 2014 Elsevier Inc. All rights reserved.
Leishmaniasis is a disease caused by protist parasites of the genus Leishmania and is transmitted by the bite of a female phlebotominesand fly. Leishmaniasis can manisfest in three different forms: cutaneous, mucocutaneous and visceral (Bero et al., 2011).
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The cutaneous form affects an estimated 10 million people today (WHO, 2007). In Tunisia, both visceral (VL) and cutaneous (CL) forms have been reported. There are three clinico-epidemiological forms of cutaneous leishmaniasis described so far: zoonotic (ZCL); sporadic (SCL) and chronic cutaneous leishmaniasis (CCL), caused respectively by Leishmania major, Leishmania infantum and Leishmania tropica (Aoun et al., 2001; Ben Ismail and Ben Rachid, 1989). Since the 1940s, the treatment for the disease consists in the administration of pentavalentantimonials and, in case of therapeutic resistance to this molecule, amphotericin B deoxycholate and liposomal amphotericin B can be used (Kayser et al., 2000). However, these drugs are considerably toxic to the patients and prone to drug resistance. Thus, a need for identifying alternative natural and safe sources of leishmanicidal molecules is urgent and the search for novel active substances, especially of plant origin, has notably increased in recent years. More recently, miltefosine was described to exhibit both in vitro and in vivo activity against Leishmania parasites (Godinho et al., 2012). Recently, many natural products have been reported to show antileishmanial activity, including naphthoquinones, lignans, neolignans, alkaloids, chalcones andtriterpenoids (Camacho et al., 2000; Delonrenzi et al., 2001; Kayser et al., 2000; Sauvain et al., 1996). The olive tree (Olea europeaea, Oleaceae) has historically provided huge economic and dietetic benefits to the Mediterranean basin (Japón-Luján and Luque de Castro, 2006). In fact Olive leaf extracts (OLE) have also been used by native people of these areas in folk medicine to treat fever and other diseases such as malaria. In the present study an attempt was made to evaluate the antioxidant and leishmanicidal activities besides presence of phytochemicals in leaf extracts of 5 different Tunisian olive varieties.
Fig. 2. Limouni variety.
2. Materials and methods 2.1. Plant material Plant materials (leaves) of five different olive tree varieties Zarrazi, Limouni (Fig. 1), Chemlali Tataouine, Dhokkar (Fig. 2), and Toffehi were collected during the maturing fruit season 2009/ 2010. All these trees were selected in the southeastern part of Tunisia Ain el Maaguel, Douiret (Fig. 3). This area is characterized by an arid Mediterranean climate, where the mean annual rainfall does not exceed 200 mm. Fig. 3. Ain el Maaguel, Douiret, Tataouine, Tunisia.
2.2. Maceration The collected olive leaves were ground to a fine powder using a mill. Each shell powder sample (0.25 g) was macerated separately
with 20 ml of extraction solvents (Ethanol, methanol, and mixture) in a capped glass tube on an agitating plate at a constant stirring rate (280 rpm) for 1 h and under 55 °C. Afterwards, a rotary vacuum evaporator at 40 °C was used in order to remove solvent. 2.3. Determination of total polyphenol contents
Fig. 1. Dhokkar variety.
Total polyphenols of olive leaf extracts were determined by Folin–Ciocalteu procedure (Škerget et al., 2005). To aliquots of 0.5 ml of diluted extracts (at a dilution of 1:10 in distilled water), 2.5 ml of Folin–Ciocalteu reagent (Sigma–Aldrich, diluted 10 times with distilled water) and 2 ml of aqueous sodium carbonate solution Na2CO3 (75 g/l) were added after 3 min. Samples were vortexed and incubated at 50 °C for 5 min and then cooled. For the blank assay, 0.5 ml of distilled water was used. The absorbance of the solutions was measured at 760 nm. The amount of total polyphenols was calculated as a gallic acid equivalent from the calibration curve of gallic acid standard solutions (0–200 lg/ml) and expressed as mg gallic acid/g dry extracts. All measurements were done in triplicate.
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2.4. Determination of total flavonoids contents Estimation of the TFC in crude extracts was performed according to the procedures described by Jia et al. (1999) and modified by Dewanto et al. (2002). Briefly, 0.25 ml of the olive leaf extracts (at a dilution of 1:10 in water) or a different concentration of the rutin standard solution was mixed with 1.25 ml of distilled water in a test tube followed by addition of 75 ll of a 5% NaNO2 solution. After 6 min, 150 ll of a 10% AlCl36H2O solution was added and allowed to stand for another 5 min before 0.5 ml of 1 M NaOH was added. The mixture was brought to 2.5 ml with distilled water and mixed with a vortex. The absorbance was measured immediately against the blank solution at 510 nm an Uvi Light XS2 spectrophotometer (Secomam, France). Samples were analyzed as triplicates. Results were expressed as mean values (milligrams of rutin equivalents per gram of dry weight) against a calibration curve of rutin (0–200 lg/ml). 3. Antioxidant activities 3.1. ABTS+ method This assay is based on decoloration that occurs when the radical cation ABTS+ is reduced to ABTS (2,20-azinobis-3-ethylbenzothiazoline-6-sulfonic acid). The radical was generated by reaction of a 7 mM solution of ABTS in water with 2.45 mM potassium persulphate (1:1). The assay was performed with 980 ll of ABTS+ solutions and 20 ll of the sample (at a dilution of 1:50 in distillated water generated with BASIC/PHQ4 Pure Hit still, India) (Rivero-Pérez et al., 2007). Absorbance measurements at 734 nm were performed after 15 min of reaction time. The results were expressed in mM equivalent Trolox/g of dry weight, using a relevant calibration curve of Trolox (0–700 lM). Samples were analyzed Triplicate.
medium. In all tests, 1% of dimethylsulfoxide (DMSO; Sigma Chemical Co., St. Louis, MO) was used to dissolve the highest dose of the compounds without affecting the parasite growth. Promastigotes were adapted for growth at 26 °C in RPMI 1640 modified medium (Gibco) and supplemented with 10% heat-inactivated fetal bovine serum. Logarithmic phase cultures were used for experimental purposes, and the in vitro susceptibility assay was performed in sterilized 96-well microtiter plates (Corning™). After that 106/well parasites were added to these wells, and the drug concentration to be tested. The final volume was 200 ll in each well. Subsequently the plates were analyzed, during an interval of time between 48 and 72 h, on a Microplate Reader Model 680 (Biorad, Hercules, CA) using a test wavelength of 570 nm and a reference wavelength of 630 nm. Percentages of inhibition, 50% inhibitory concentrations (IC50) for active compounds were calculated by linear regression analysis with 95% confidence limits. All experiments were performed three times each in duplicate and the mean values were also calculated. A paired two-tailed t-test was used for analysis of the data. Values of p < 0.05 were considered significant. The obtained inhibition curves statistical analysis was undertaken using the Sigma Plot 12.0 software program (Systat Software Inc.). 5. Statistical analysis The results were expressed as mean ± standard deviation. Analysis of variance was determined by one-way ANOVA using STATGRAPHICS Centurion XVI. Differences at p < 0.05 were considered statistically significant. The IC50 was performed with the program Sigmaplot12. 6. Results and discussion 6.1. Total phenol, flavonoids content and antioxidant activity
3.2. DPPH method This method is based on the reduction of the free radical DPPH (2,2-diphenyl-1-picrylhydrazyl) (Brand-Williams et al., 1995). The reaction takes place when 980 ll of DPPH. (60 lM) was mixed with 20 ll of the diluted sample. After a reaction time of 2 h absorbance values at 517 nm were measured. The results were expressed in mM equivalent Trolox/g of dry weight, using a relevant calibration curve of Trolox (0–700 lM). Samples were analyzed Triplicate. 4. Leishmanicidal activity 4.1. Parasite strain The activity of the collected material was evaluated against promastigotes of Leishmania donovani (MHOM/IN/90/GE1F8R), L. tropica (MON 58/LEM 2578), L. major (LM 252) and L. amazonensis (MHOM/BR/77/LTB0016). 4.2. In vitro effect against the promastigotes stage of Leishmania spp. The leishmanicidal activities of the assayed drugs were determined by the modified Alamar BlueÒ assay as described by Cabrera-Serra et al. (2007). This method is based on anoxido/ reduction reaction. In presence of viable cells, the blue, nonfluorescent, oxidized form of the resazurin (principal ingredient) becomes pink and fluorescent upon reduction. In fact, this reaction could be measure with a fluorometer or spectrophotometer (O’Brien et al., 2000). For the in vitro study, samples were dissolved in dimethylsulfoxide (DMSO) and further dilutions were made with RPMI 1640
The total phenols content of the extracts was different according to the variety. Limouni showed the high amount of these compounds, with 153.917 ± 0.528 mg GAE/g of dry extract, being 2.65-fold higher than the Zarrazi variety. The lowest amounts were obtained for Zarrazi methanolic extract with 46.809 mg GAE/g of dry extract. The content of total flavonoids varied significantly among species. Toffehi methanolic extracts had the highest content of flavonoids (42.754 ± 0.674 mg RE/g of dry extract), followed by Limouni methanolic extract (36.450 ± 1.079 mg RE/g of dry extract). Zarrazi ethanolic extract had the lowest content of the total flavonoids, 18.397 mg RE/g of dry extracts, among the five cultivars (Table 1). Average antioxidant capacities for the cultivars in this study were determined using two methods: the DPPH and ABTS+ (Table 1). The DPPH free-radical scavenging method incorporates a metastable free radical that is capable of accepting hydrogen radicals from antioxidants in solution. Although DPPH. is a deep purple in solution, it becomes colòrless in its reduced form, so the reaction between DPPH. and an antioxidant can be monitored by the decrease in absorbance of the colored free radical (BrandWilliams et al., 1995). In the DPPH radical scavenging activity assay, the Toffehi mixture extracts exhibited the highest antioxidant activity (0.492 M TE/g of dry extracts). In other case, for the ABTS radical scavenging assay, the Limouni ethanolic extracts had high antioxidant potential (0.251 M TE/g of dry extracts). In general, most phenolics and flavonoids exhibit some degree of antioxidant activity. Accordingly, the extract with strong antioxidant activity would generally present the higher content of phenolics or flavonoids. Different reports are found from the literatures, whereby some authors suggested correlation between all these
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I. Sifaoui et al. / Experimental Parasitology 141 (2014) 106–111 Table 1 Phytochemical analysis and antioxidant activity of the evaluated olive leaf extracts. Variety Phenols mg EAG/g dry extract
Flavonoids mg ER/g dry extract
ABTS M Trolox/g dry extract
DPPH M Trolox/g dry extract
Ethanol Methanol Mixture Ethanol Methanol Mixture Ethanol Methanol Mixture Ethanol Methanol Mixture
Limouni
Zarrazi
Dhokkar
Toffehi
Chemlali Tataouine
134.156 ± 1.155 aB 153.917 ± 0.528 bA 125.271 ± 0.759 bC 35.161 ± 0.000 bBC 36.450 ± 1.079 cAB 36.880 ± 0.337 bA 0.198 ± 0.003 bC 0.251 ± 0.008 aA 0.218 ± 0.002 aB 0.401 ± 0.033 bC 0.540 ± 0.020 bA 0.494 ± 0.006 bB
46.809 ± 1.038 fB 58.000 ± 0.322 fA 45.139 ± 0.107 fC 19.257 ± 0.072 eA 18.397 ± 0.143 fB 19.257 ± 0.358 eA 0.163 ± 0.003 dB 0.184 ± 0.000 dA 0.156 ± 0.009 eB 0.107 ± 0.004 eNS 0.100 ± 0.003 eNS 0.116 ± 0.012 eNS
50.807 ± 1.106 eC 61.029 ± 0.474 eA 56.070 ± 0.253 eB 22.839 ± 0.38 dB 32.295 ± 1.835 dA 22.695 ± 0.886 dB 0.177 ± 0.003 cC 0.209 ± 0.001 cA 0.191 ± 0.003 cdB 0.284 ± 0.023 dB 0.336 ± 0.017 dA 0.308 ± 0.017 dAB
54.552 ± 0.101 dC 74.946 ± 0.673 dA 60.204 ± 0.336 dB 21.692 ± 0.674 dC 42.754 ± 0.674 bA 26.850 ± 0.472 cB 0.180 ± 0.009 cB 0.225 ± 0.001 bA 0.190 ± 0.009 dB 0.344 ± 0.022 cC 0.441 ± 0.012 cA 0.402 ± 0.021 cB
64.116 ± 0.256 cB 77.622 ± 1.230 cA 64.470 ± 0.568 cB 27.280 ± 1.033 cA 25.274 ± 0.265 eAB 25.704 ± 0.895 cB 0.195 ± 0.002 bB 0.211 ± 0004 cA 0.200 ± 0.003 bcB 0.341 ± 0.005 cC 0.442 ± 0.006 cA 0.412 ± 0.002 cB
⁄ Mean ± standard deviation. a–b, means within a column row with different letters are significantly different (p < 0.05). A–B, means within a row different letters are significantly different (p < 0.05). NS, non-significant.
Table 2 Correlation coefficients between the phytochemical content and the antioxidant activities. Assay
Phenols
Flavonoids
DPPH ABTS
0.932 0.736
0.529 0.583
parameters (Pyo et al., 2004; Sun and Ho, 2006), while others found no relationship. Consistent with some work, total phenols values were highly correlated with DPPH and ABTS antioxidant values with R2 values of 0.932 and 0.736, respectively (p < 0.001). However, moderate to weak correlation was found between the flavonoids content and both antioxidant values with a regression coefficient R2 less than 0.580 (p < 0.05). Many previous reports confirm those results, Andarwulan et al. (2010) have showing that the content of flavonoid present on fresh vegetables from Indonesia do not correlate with the antioxidant activity measured with DPPH and ABTS+ (Table 2). The phenol and flavonoids content as well as the antioxidant activity measured with both method DPPH and ABTS+ were significantly affected by the olive cultivar with p respectively 0.002, 0.000, 0.002, and 0.001. 6.2. Leishmanicidal activity of olive leaf extracts In the present study, olive leaf extracts from different varieties and macerate with three solvent were screened for their activity
against four Leishmania strains. IC50 values of different extracts on Leishmania are summarized on Table 3. Parasites were separately incubated with different extracts concentrations for 48 h at 24 °C. In the evaluation of the results, it could be observed that the leishmanicidal activity was based on a dependent-dose application. The antiparasitic activity was significantly affected by the olive cultivar and the Leishmania strains tested with p respectively 0.0057 and 0.000. Meanwhile, the effect of the solvent was moderate to not significant on the activity (p = 0.0443). Indeed, both solvents are characterized by a high polarity; consequently, the phytochemical profile extract by them could be the same. Extraction with solvents characterized by different polarity would be more interesting. Second order ANOVA analysis revealed that the Limouni cultivar was the most active extract against all test strains, in other hand Leishmania amazonensis seems to be the most sensitive strain to olive leaf extract with an IC50 < 17.926 ± 1.876 lg/ml (Fig. 4). For L. donovani the IC50 ranged from 2.130 ± 0.023 to 71.570 ± 4.324 lg/ml for the ethanolic extract of Limouni and Zarrazi variety, respectively. The IC50 of the Limouni variety against L. tropica and L. major are respectively, 17.622 and 14.660 lg/ml. Consequently this variety is the stronger inhibitor of these strains. In fact, this activity could be correlated to the amount of phenolic compounds in this cultivar. Compared to the results found by Kyriazis et al. (2013), alcoholic extracts exhibit a stronger activity than aqueous extracts. Indeed, they report that the IC50 of OLWE (olive leaf water extraction) was 60.000 ± 9.5 lg/ml against L. donovani MON-2 promastigotes. Kyriazis et al. (2013) reported that both oleuropein and hydroxytyrosol exhibit a good activity against
Table 3 Leishmanicidal activity of the evaluated olive leaf extracts (IC50 on lg of dry extract/ml). Variety L. amazonensis
L. donovani
L. tropica
L. major
Limouni Ethanol Methanol Mixture Ethanol Methanol Mixture Ethanol Methanol Mixture Ethanol Methanol Mixture
12.353 ± 1.041 6.970 ± 0.316 12.353 ± 0.366 11.234 ± 1.382 2.130 ± 0.023 25.220 ± 3.113 23.614 ± 1.515 17.622 ± 0.651 23.730 ± 1.262 25.613 ± 1.440 14.661 ± 0.380 29.300 ± 2.480
cB aA cB aB aA bC aB bA bB bB aA bC
Zarrazi
Dhokkar
Toffehi
14.379 ± 1.400 cdA 16.950 ± 1.311 dAB 17.926 ± 1.876 dB 17.737 ± 1.430 cA 71.570 ± 4.324 fC 46.533 ± 1.582 dB 46.311 ± 3.228 dB 27.707 ± 2.852 cA 48.942 ± 4.183 dB 54.301 ± 2.260 dB 23.889 ± 1.651 cA 57.293 ± 3.945 dB
4.233 ± 0.023 aB 7.841 ± 0.611 abC 2.936 ± 0.021 aA 16.296 ± 0.63 bcA 26.435 ± 0.843 cB 25.546 ± 1.102 bB 44.011 ± 2.632 dB 19.494 ± 0.785 bA 46.648 ± 1.373 dB 55.734 ± 2.753 dB 23.120 ± 1.581 cA 58.954 ± 0.740 dB
4.017 ± 1.345 8.018 ± 1.657 5.842 ± 0.755 13.785 ± 0.976 56.946 ± 1.265 37.245 ± 1.560 35.612 ± 1.537 17.761 ± 0.860 33.794 ± 2.205 42.159 ± 2.777 17.681 ± 1.383 36.876 ± 1.211
⁄ Mean ± standard deviation. a–b, means within a column row with different letters are significantly different (p < 0.05). A–B, means within a row different letters are significantly different (p < 0.05). NS, non-significant.
Chemlali Tataouine aA abB bAB abA eC cB cB bA cB cC bA cB
16.199 ± 2.020 dC 9.277 ± 1.155 bcB 3.894 ± 0.599 aA 53.8818 ± 3.108 eC 6.201 ± 0.939 bA 14.529 ± 3.250aB 31.653 ± 1.779 bB 18.875 ± 1.253 bA 32.295 ± 2.191 cB 39.506 ± 3.146 cC 17.323 ± 1.066 bA 32.208 ± 1.930 bB
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Fig. 4. Effect of the methanolic extract of Limouni variety on Leishmania amazonensis at 48 h (25 lg/ml) (a), negative control (b).
L. infantum and L. donovani. The relative leishmanicidal activity of the five varieties against L. tropica and L. major, was moderately correlated to phenols content, with a regression coefficient respectively of 0.762 and 0.742 (p < 0.001). The flavonoids content of the different extracts was correlated with the leishmanicidal activity against two L. tropica and major (R2L: Tropica ¼ 0:846 and R2L: Major ¼ 0:777, p < 0.001). Flavonoids are broad classes of plant phenolics that are known to possess a well-established protective effect against membrane lipoperoxidative damages and have long been under investigation for antiparasitic activity (Sen et al., 2004). Mendonça-Filho et al. (2004) have revealed the leishmanicidal activity of polyphenolic-rich extracts from husk fiber of Cocosnucifera Linn. (Palmae). The correlation analysis revealed the absence of correlation between the phenolic compounds especially the flavonoid and the leishmanicidal activity against L. donovani and Leishmania amazonensis, suggesting the presence of other bioactive compounds. The olive leaf are rich in polyphenolic compounds such hydroxytyrosol, tyrosol, benzoic acids, in secoiridoids like the oleuropein, in flavonoids and in triterpenoid such as oleanolic acid, maslinic acid, and ursolic acid (Papoti and Tsimidou, 2009). Several studies, proclaimed the antileishmanial activity of triterpenic acid. Torres-Santos et al. (2004), showed that oleanolic acid and ursolic acid, triterpenic acids isolated from the methanolic fraction of Pourouma guianensis exhibit a strong activity against Leishmania amazonensis. 7. Conclusion Despite the fact that the four Leishmania species tested in this study are able to cause different forms of leishmaniasis, the olive leaf extracts evaluated in this work seems to possess interesting leishmanicidal properties against all of them. Furthermore, this activity could be the result of the presence of several molecules especially phenolic compounds. These results suggest that there could be a potential for new compounds to develop new drugs against leishmaniasis. However, bio guided purification will be necessary to isolate and purified the bioactive compounds from olive leaf extract and further evaluate these observations. Acknowledgments This work was supported by the Grants RICET (project No. RD06/0021/0005 of the programme of Redes Temáticas de Investigación Cooperativa, FIS), Spanish Ministry of Health, Madrid, Spain and the Project FIS PI10/01298 ‘‘Protozoosis emergentes por amebas de vida libre: aislamiento y caracterización molecular, identificación de cepas transportadoras de otros agentes patógenos y búsqueda de quimioterapias efectivas’’ and PI13/00490 ‘‘Protozoosis Emergentes por Amebas de Vida Libre: Aislamiento, Caracterización, Nuevas Aproximaciones Terapéuticas y Traslación
Clínica de los Resultados’’ from the Instituto de Salud Carlos III. I.S. was funded by an alternating Scholarship from the University of Carthage, Tunisian Ministry of Higher Education and Scientific Research. A.L.A. was funded by a Grant ‘‘Ayudas del Programa de Formación de Personal Investigador, para la realización de Tesis Doctorales’’ from the Agencia Canaria de Investigación, Innovación y Sociedad de la Información from the Canary Islands Government. M.R.B. was funded by CEI Canarias, Campus Atlántico Internacional and Becas Fundación Cajacanarias para Postgraduados 2014. C.M.M.N. was supported by a postdoctoral Grant from the Fundación Canaria Manuel Morales, La Palma, Canary Islands. J.L.M. was supported by the Ramón y Cajal Subprogramme from the Spanish Ministry of Science and Innovation RYC-2011-08863. References Andarwulan, N., Batari, R., Sandrasari, D.A., Bolling, B., Wijay, H., 2010. Flavonoid content and antioxidant activity of vegetables from Indonesia. Food Chem. 121, 1231–1235. Aoun, K., Bouratbine, A., Harrat, Z., Belkaid, M., BelHadj Ali, S., 2001. Particular profile of the zymodemes of Leishmania infantum causing visceral leishmaniasis in Tunisia. Bull. Soc. Pathol. Exot. 94, 375–377. Ben Ismail, R., Ben Rachid, R., 1989. Epidémiologie des leishmanioses en Tunisie. In: Maladies Tropicales Transmissibles. AUPELF UREF, John Libbey Eurotex, Paris, pp. 73–80. Bero, J., Hannaert, V., Chataigné, G., Hérent, M.F., Quetin-Leclercq, J., 2011. In vitro antitrypanosomal and antileishmanial activity of plants used in Benin in traditional medicine and bio-guided fractionation of the most active extract. J. Ethnopharmacol. 137, 998–1002. Brand-Williams, W., Cuvelier, M.E., Berset, C., 1995. Use of a free radical method to evaluate antioxidant activity. LWT – Food Sci. Technol. 28, 25–30. Cabrera-Serra, M.G., Lorenzo-Morales, J., Romero, M.L., Valladares, B., Piñero, J.E., 2007. In vitro activity of perifosine: a novel alkylphospholipid against the promastigote stage of Leishmania species. Parasitol. Res. 100, 1155–1157. Camacho, M.D., Mata, R., Castaneda, P., Kirby, G.C., Warhurst, D.C., Croft, S.L., Phillipson, J.D., 2000. Bioactive compounds from Celaenodendron mexicanum. Planta Med. 66, 463–468. Delonrenzi, J.C., Attias, M., Gattas, C.R., Andrade, M., Rezende, C., Pinto, A.D., Henriques, A.T., Bou-Habib, D.C., Saraiva, E.M.B., 2001. Antileishmanial activity of an indole alkaloid from Peschieraaustralis. Antimicrob. Agents Chemother. 45, 1349–1354. Dewanto, V., Wu, X., Adom, K., Liu, R.H., 2002. Thermal processing enhances the nutritional value of tomatoes by increasing total antioxidant activity. J. Agric. Food Chem. 50, 3010–3014. Godinho, J.L., Simas-Rodrigues, C., Silva, R., Ürmenyi, T.P., de Souza, W., Rodrigues, J.C., 2012. Efficacy of miltefosine treatment in Leishmania amazonensis-infected BALB/c mice. Int. J. Antimicrobial. Agents 39, 326–331. Japón-Luján, R., Luque de Castro, M.D., 2006. Superheated liquid extraction of oleuropein and related biophenols from olive leaf. J. Chromatogr. A 1136, 185– 191. Jia, Z.S., Tang, M.C., Wu, J.M., 1999. The determination of flavonoid contents in mulverry and their scavenging effects on superoxide radicals. Food Chem. 64, 555–559. Kayser, O., Kiderlen, A.F., Laatsch, H., Croft, S.L., 2000. In vitro leishmanicidal activity of monomeric and dimeric naphthoquinones. Acta Trop. 77, 307–314. Kyriazis, J.D., Aligiannis, N., Polychronopoulos, P., Skaltsounis, A.L., Dotsika, E., 2013. Leishmanicidal activity assessment of olive tree extracts. Phytomedicine 20, 275–281. Mendonça-Filho, R.R., Rodrigues, I.A., Alviano, D.S., Santos, A.L., Soares, R.M., Alviano, C.S., Lopes, A.H., Rosa, Mdo S., 2004. Leishmanicidal activity of polyphenolic-rich extracts from husk fiber of Cocosnucifera Linn. (Palmae). Res. Microbiol. 155, 136–143.
I. Sifaoui et al. / Experimental Parasitology 141 (2014) 106–111 O’Brien, J., Wilson, I., Orton, T., Pognan, F., 2000. Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur. J. Biochem. 267, 5421–5426. Papoti, V., Tsimidou, M.Z., 2009. Impact of sampling parameters on the radical scavenging potential of olive (Olea europaea L.) leaf. J. Agric. Food Chem. 57, 3470–3477. Pyo, Y.H., Lee, T.C., Logendra, L., Rosen, R.T., 2004. Antioxidant activity and phenolic compounds of Swiss chard. Food Chem. 85, 19–26. Rivero-Pérez, M.D., Muñiz, P., González-Sanjosé, M.L., 2007. Antioxidant profile of red wines evaluated by total antioxidant capacity, scavenger activity and biomarkers of oxidative stress methodologies. J. Agric. Food Chem. 55, 5476–5783. Sauvain, M., Kunesch, N., Poisson, J., Gantier, J.C., Gayral, P., Dedet, J.P., 1996. Isolation of leishmanicidal triterpenes and lignans from the Amazonian liana Doliocarpus dentatus (Dilleniaceae). Phytother. Res. 10, 1–4.
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Sen, G., Mukhopadhaya, R., Ghosal, J., Biswas, T., 2004. Combination of ascorbate and a-tocopherol as a preventive therapy against structural and functional defects of erythrocytes in visceral leishmaniasis. Free Radic. Res. 38, 527– 534. Škerget, M., Kotnik, P., Hadolin, M., Hraš, A.R., Simonicˇ, M., Knez, Zˇ., 2005. Phenols, proanthocyanidins, flavones and flavonols in some plant materials and their antioxidant activities. Food Chem. 89, 191–198. Sun, T., Ho, C.T., 2006. Antioxidant activities of buckwheat extracts. Food Chem. 90, 743–749. Torres-Santos, E.S., Lopes, D., Rodrigues Oliveira, R., Carauta, J.P.P., Bandeira Falcao, C.A., Kaplan, M.A.C., Rossi-Bergmann, B., 2004. Antileishmanial activity of isolated triterpenoids from Pourouma guianensis. Phytomedicine 11, 114–120. World Health Organisation (WHO), 2007. WHO/CDS/NTD/IDM/2007.3. Cutaneous leishmaniasis. Why are you neglecting me?.
Contents lists available at ScienceDirect
Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr
Activity of olive leaf extracts against the promastigote stage of Leishmania species and their correlation with the antioxidant activity Ines Sifaoui a,⇑, Atteneri López-Arencibia b,1, Carmen Ma Martín-Navarro b,c,1, Nadia Chammem d,2, María Reyes-Batlle b,1, Mondher Mejri a,3, Jacob Lorenzo-Morales b,1, Manef Abderabba a,3, José E. Piñero b,1 a
Laboratoire Matériaux-Molécules et Applications, IPEST, University of Carthage, B.P. 51, 2070 La Marsa, Tunisia University Institute of Tropical Diseases and Public Health, University of La Laguna, Avda Francisco Sanchez s/n, Campus de Anchieta, 38271 La Laguna, Tenerife, Canary Islands, Spain Centre for Integrative Physiology, University of Edinburgh, Scotland, UK d Laboratoire d’Ecologie et de Technologie Microbienne, INSAT, University of Carthage, Centre Urbain Nord, B.P. 676, 1080 Tunis Cedex, Tunisia b c
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Olive leaf extract present an
interesting leishmanicidal activity. The antiparasitic activity was
significantly affected by the olive cultivar and the tested Leishmania strains. The Limouni variety presented the highest content on phenolic compounds and the stronger leishmanicidal activity. L. amazonensis was the most sensitive strain to the assayed olive leaf extract.
a r t i c l e
i n f o
Article history: Received 17 April 2013 Received in revised form 19 February 2014 Accepted 4 March 2014 Available online 21 March 2014 Keywords: Olive leaf extract Leishmanicidal activity Antioxidant activity
a b s t r a c t Leishmaniasis is one of the neglected tropical diseases in terms of drug discovery and development. Furthermore, the chemotherapy used to treat this disease has been proved to be highly toxic and to present resistance issues. As consequent, the need for novel leishmanicidal molecules has notably increased in the recent years. In the present work an attempt was made to evaluate the antioxidant and leishmanicidal activities besides presence of compounds in leaf extracts of 5 different Tunisian olive tree varieties, used as traditional medicine in this country. The concentration of extracts needed to inhibit 50% of the parasitic growth (IC50) was estimated using different Leishmania strains. All tested extracts showed an inhibitory effect on the parasite growth with IC50 ranging from 2.130 ± 0.023 to 71.570 ± 4.324 lg/ml, respectively for the methanolic extracts of Limouni and Zarrazi against Leishmania donovani. In fact, this activity was significantly affected by the olive cultivar and the tested Leishmania strain. Furthermore, the activities against both Leishmania tropica and major species were correlated to the total phenolic compounds. These results could suggest that olive leaf extract could carry potential new compounds for the development of novel drugs against Leishmaniasis. Ó 2014 Elsevier Inc. All rights reserved.
1. Introduction ⇑ Corresponding author. Fax: +216 71 746 551. 1 2 3
E-mail address: [email protected] (I. Sifaoui). Fax: +34 922318514. Fax: +216 71704329. Fax: +216 71746551.
http://dx.doi.org/10.1016/j.exppara.2014.03.002 0014-4894/Ó 2014 Elsevier Inc. All rights reserved.
Leishmaniasis is a disease caused by protist parasites of the genus Leishmania and is transmitted by the bite of a female phlebotominesand fly. Leishmaniasis can manisfest in three different forms: cutaneous, mucocutaneous and visceral (Bero et al., 2011).
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The cutaneous form affects an estimated 10 million people today (WHO, 2007). In Tunisia, both visceral (VL) and cutaneous (CL) forms have been reported. There are three clinico-epidemiological forms of cutaneous leishmaniasis described so far: zoonotic (ZCL); sporadic (SCL) and chronic cutaneous leishmaniasis (CCL), caused respectively by Leishmania major, Leishmania infantum and Leishmania tropica (Aoun et al., 2001; Ben Ismail and Ben Rachid, 1989). Since the 1940s, the treatment for the disease consists in the administration of pentavalentantimonials and, in case of therapeutic resistance to this molecule, amphotericin B deoxycholate and liposomal amphotericin B can be used (Kayser et al., 2000). However, these drugs are considerably toxic to the patients and prone to drug resistance. Thus, a need for identifying alternative natural and safe sources of leishmanicidal molecules is urgent and the search for novel active substances, especially of plant origin, has notably increased in recent years. More recently, miltefosine was described to exhibit both in vitro and in vivo activity against Leishmania parasites (Godinho et al., 2012). Recently, many natural products have been reported to show antileishmanial activity, including naphthoquinones, lignans, neolignans, alkaloids, chalcones andtriterpenoids (Camacho et al., 2000; Delonrenzi et al., 2001; Kayser et al., 2000; Sauvain et al., 1996). The olive tree (Olea europeaea, Oleaceae) has historically provided huge economic and dietetic benefits to the Mediterranean basin (Japón-Luján and Luque de Castro, 2006). In fact Olive leaf extracts (OLE) have also been used by native people of these areas in folk medicine to treat fever and other diseases such as malaria. In the present study an attempt was made to evaluate the antioxidant and leishmanicidal activities besides presence of phytochemicals in leaf extracts of 5 different Tunisian olive varieties.
Fig. 2. Limouni variety.
2. Materials and methods 2.1. Plant material Plant materials (leaves) of five different olive tree varieties Zarrazi, Limouni (Fig. 1), Chemlali Tataouine, Dhokkar (Fig. 2), and Toffehi were collected during the maturing fruit season 2009/ 2010. All these trees were selected in the southeastern part of Tunisia Ain el Maaguel, Douiret (Fig. 3). This area is characterized by an arid Mediterranean climate, where the mean annual rainfall does not exceed 200 mm. Fig. 3. Ain el Maaguel, Douiret, Tataouine, Tunisia.
2.2. Maceration The collected olive leaves were ground to a fine powder using a mill. Each shell powder sample (0.25 g) was macerated separately
with 20 ml of extraction solvents (Ethanol, methanol, and mixture) in a capped glass tube on an agitating plate at a constant stirring rate (280 rpm) for 1 h and under 55 °C. Afterwards, a rotary vacuum evaporator at 40 °C was used in order to remove solvent. 2.3. Determination of total polyphenol contents
Fig. 1. Dhokkar variety.
Total polyphenols of olive leaf extracts were determined by Folin–Ciocalteu procedure (Škerget et al., 2005). To aliquots of 0.5 ml of diluted extracts (at a dilution of 1:10 in distilled water), 2.5 ml of Folin–Ciocalteu reagent (Sigma–Aldrich, diluted 10 times with distilled water) and 2 ml of aqueous sodium carbonate solution Na2CO3 (75 g/l) were added after 3 min. Samples were vortexed and incubated at 50 °C for 5 min and then cooled. For the blank assay, 0.5 ml of distilled water was used. The absorbance of the solutions was measured at 760 nm. The amount of total polyphenols was calculated as a gallic acid equivalent from the calibration curve of gallic acid standard solutions (0–200 lg/ml) and expressed as mg gallic acid/g dry extracts. All measurements were done in triplicate.
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2.4. Determination of total flavonoids contents Estimation of the TFC in crude extracts was performed according to the procedures described by Jia et al. (1999) and modified by Dewanto et al. (2002). Briefly, 0.25 ml of the olive leaf extracts (at a dilution of 1:10 in water) or a different concentration of the rutin standard solution was mixed with 1.25 ml of distilled water in a test tube followed by addition of 75 ll of a 5% NaNO2 solution. After 6 min, 150 ll of a 10% AlCl36H2O solution was added and allowed to stand for another 5 min before 0.5 ml of 1 M NaOH was added. The mixture was brought to 2.5 ml with distilled water and mixed with a vortex. The absorbance was measured immediately against the blank solution at 510 nm an Uvi Light XS2 spectrophotometer (Secomam, France). Samples were analyzed as triplicates. Results were expressed as mean values (milligrams of rutin equivalents per gram of dry weight) against a calibration curve of rutin (0–200 lg/ml). 3. Antioxidant activities 3.1. ABTS+ method This assay is based on decoloration that occurs when the radical cation ABTS+ is reduced to ABTS (2,20-azinobis-3-ethylbenzothiazoline-6-sulfonic acid). The radical was generated by reaction of a 7 mM solution of ABTS in water with 2.45 mM potassium persulphate (1:1). The assay was performed with 980 ll of ABTS+ solutions and 20 ll of the sample (at a dilution of 1:50 in distillated water generated with BASIC/PHQ4 Pure Hit still, India) (Rivero-Pérez et al., 2007). Absorbance measurements at 734 nm were performed after 15 min of reaction time. The results were expressed in mM equivalent Trolox/g of dry weight, using a relevant calibration curve of Trolox (0–700 lM). Samples were analyzed Triplicate.
medium. In all tests, 1% of dimethylsulfoxide (DMSO; Sigma Chemical Co., St. Louis, MO) was used to dissolve the highest dose of the compounds without affecting the parasite growth. Promastigotes were adapted for growth at 26 °C in RPMI 1640 modified medium (Gibco) and supplemented with 10% heat-inactivated fetal bovine serum. Logarithmic phase cultures were used for experimental purposes, and the in vitro susceptibility assay was performed in sterilized 96-well microtiter plates (Corning™). After that 106/well parasites were added to these wells, and the drug concentration to be tested. The final volume was 200 ll in each well. Subsequently the plates were analyzed, during an interval of time between 48 and 72 h, on a Microplate Reader Model 680 (Biorad, Hercules, CA) using a test wavelength of 570 nm and a reference wavelength of 630 nm. Percentages of inhibition, 50% inhibitory concentrations (IC50) for active compounds were calculated by linear regression analysis with 95% confidence limits. All experiments were performed three times each in duplicate and the mean values were also calculated. A paired two-tailed t-test was used for analysis of the data. Values of p < 0.05 were considered significant. The obtained inhibition curves statistical analysis was undertaken using the Sigma Plot 12.0 software program (Systat Software Inc.). 5. Statistical analysis The results were expressed as mean ± standard deviation. Analysis of variance was determined by one-way ANOVA using STATGRAPHICS Centurion XVI. Differences at p < 0.05 were considered statistically significant. The IC50 was performed with the program Sigmaplot12. 6. Results and discussion 6.1. Total phenol, flavonoids content and antioxidant activity
3.2. DPPH method This method is based on the reduction of the free radical DPPH (2,2-diphenyl-1-picrylhydrazyl) (Brand-Williams et al., 1995). The reaction takes place when 980 ll of DPPH. (60 lM) was mixed with 20 ll of the diluted sample. After a reaction time of 2 h absorbance values at 517 nm were measured. The results were expressed in mM equivalent Trolox/g of dry weight, using a relevant calibration curve of Trolox (0–700 lM). Samples were analyzed Triplicate. 4. Leishmanicidal activity 4.1. Parasite strain The activity of the collected material was evaluated against promastigotes of Leishmania donovani (MHOM/IN/90/GE1F8R), L. tropica (MON 58/LEM 2578), L. major (LM 252) and L. amazonensis (MHOM/BR/77/LTB0016). 4.2. In vitro effect against the promastigotes stage of Leishmania spp. The leishmanicidal activities of the assayed drugs were determined by the modified Alamar BlueÒ assay as described by Cabrera-Serra et al. (2007). This method is based on anoxido/ reduction reaction. In presence of viable cells, the blue, nonfluorescent, oxidized form of the resazurin (principal ingredient) becomes pink and fluorescent upon reduction. In fact, this reaction could be measure with a fluorometer or spectrophotometer (O’Brien et al., 2000). For the in vitro study, samples were dissolved in dimethylsulfoxide (DMSO) and further dilutions were made with RPMI 1640
The total phenols content of the extracts was different according to the variety. Limouni showed the high amount of these compounds, with 153.917 ± 0.528 mg GAE/g of dry extract, being 2.65-fold higher than the Zarrazi variety. The lowest amounts were obtained for Zarrazi methanolic extract with 46.809 mg GAE/g of dry extract. The content of total flavonoids varied significantly among species. Toffehi methanolic extracts had the highest content of flavonoids (42.754 ± 0.674 mg RE/g of dry extract), followed by Limouni methanolic extract (36.450 ± 1.079 mg RE/g of dry extract). Zarrazi ethanolic extract had the lowest content of the total flavonoids, 18.397 mg RE/g of dry extracts, among the five cultivars (Table 1). Average antioxidant capacities for the cultivars in this study were determined using two methods: the DPPH and ABTS+ (Table 1). The DPPH free-radical scavenging method incorporates a metastable free radical that is capable of accepting hydrogen radicals from antioxidants in solution. Although DPPH. is a deep purple in solution, it becomes colòrless in its reduced form, so the reaction between DPPH. and an antioxidant can be monitored by the decrease in absorbance of the colored free radical (BrandWilliams et al., 1995). In the DPPH radical scavenging activity assay, the Toffehi mixture extracts exhibited the highest antioxidant activity (0.492 M TE/g of dry extracts). In other case, for the ABTS radical scavenging assay, the Limouni ethanolic extracts had high antioxidant potential (0.251 M TE/g of dry extracts). In general, most phenolics and flavonoids exhibit some degree of antioxidant activity. Accordingly, the extract with strong antioxidant activity would generally present the higher content of phenolics or flavonoids. Different reports are found from the literatures, whereby some authors suggested correlation between all these
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I. Sifaoui et al. / Experimental Parasitology 141 (2014) 106–111 Table 1 Phytochemical analysis and antioxidant activity of the evaluated olive leaf extracts. Variety Phenols mg EAG/g dry extract
Flavonoids mg ER/g dry extract
ABTS M Trolox/g dry extract
DPPH M Trolox/g dry extract
Ethanol Methanol Mixture Ethanol Methanol Mixture Ethanol Methanol Mixture Ethanol Methanol Mixture
Limouni
Zarrazi
Dhokkar
Toffehi
Chemlali Tataouine
134.156 ± 1.155 aB 153.917 ± 0.528 bA 125.271 ± 0.759 bC 35.161 ± 0.000 bBC 36.450 ± 1.079 cAB 36.880 ± 0.337 bA 0.198 ± 0.003 bC 0.251 ± 0.008 aA 0.218 ± 0.002 aB 0.401 ± 0.033 bC 0.540 ± 0.020 bA 0.494 ± 0.006 bB
46.809 ± 1.038 fB 58.000 ± 0.322 fA 45.139 ± 0.107 fC 19.257 ± 0.072 eA 18.397 ± 0.143 fB 19.257 ± 0.358 eA 0.163 ± 0.003 dB 0.184 ± 0.000 dA 0.156 ± 0.009 eB 0.107 ± 0.004 eNS 0.100 ± 0.003 eNS 0.116 ± 0.012 eNS
50.807 ± 1.106 eC 61.029 ± 0.474 eA 56.070 ± 0.253 eB 22.839 ± 0.38 dB 32.295 ± 1.835 dA 22.695 ± 0.886 dB 0.177 ± 0.003 cC 0.209 ± 0.001 cA 0.191 ± 0.003 cdB 0.284 ± 0.023 dB 0.336 ± 0.017 dA 0.308 ± 0.017 dAB
54.552 ± 0.101 dC 74.946 ± 0.673 dA 60.204 ± 0.336 dB 21.692 ± 0.674 dC 42.754 ± 0.674 bA 26.850 ± 0.472 cB 0.180 ± 0.009 cB 0.225 ± 0.001 bA 0.190 ± 0.009 dB 0.344 ± 0.022 cC 0.441 ± 0.012 cA 0.402 ± 0.021 cB
64.116 ± 0.256 cB 77.622 ± 1.230 cA 64.470 ± 0.568 cB 27.280 ± 1.033 cA 25.274 ± 0.265 eAB 25.704 ± 0.895 cB 0.195 ± 0.002 bB 0.211 ± 0004 cA 0.200 ± 0.003 bcB 0.341 ± 0.005 cC 0.442 ± 0.006 cA 0.412 ± 0.002 cB
⁄ Mean ± standard deviation. a–b, means within a column row with different letters are significantly different (p < 0.05). A–B, means within a row different letters are significantly different (p < 0.05). NS, non-significant.
Table 2 Correlation coefficients between the phytochemical content and the antioxidant activities. Assay
Phenols
Flavonoids
DPPH ABTS
0.932 0.736
0.529 0.583
parameters (Pyo et al., 2004; Sun and Ho, 2006), while others found no relationship. Consistent with some work, total phenols values were highly correlated with DPPH and ABTS antioxidant values with R2 values of 0.932 and 0.736, respectively (p < 0.001). However, moderate to weak correlation was found between the flavonoids content and both antioxidant values with a regression coefficient R2 less than 0.580 (p < 0.05). Many previous reports confirm those results, Andarwulan et al. (2010) have showing that the content of flavonoid present on fresh vegetables from Indonesia do not correlate with the antioxidant activity measured with DPPH and ABTS+ (Table 2). The phenol and flavonoids content as well as the antioxidant activity measured with both method DPPH and ABTS+ were significantly affected by the olive cultivar with p respectively 0.002, 0.000, 0.002, and 0.001. 6.2. Leishmanicidal activity of olive leaf extracts In the present study, olive leaf extracts from different varieties and macerate with three solvent were screened for their activity
against four Leishmania strains. IC50 values of different extracts on Leishmania are summarized on Table 3. Parasites were separately incubated with different extracts concentrations for 48 h at 24 °C. In the evaluation of the results, it could be observed that the leishmanicidal activity was based on a dependent-dose application. The antiparasitic activity was significantly affected by the olive cultivar and the Leishmania strains tested with p respectively 0.0057 and 0.000. Meanwhile, the effect of the solvent was moderate to not significant on the activity (p = 0.0443). Indeed, both solvents are characterized by a high polarity; consequently, the phytochemical profile extract by them could be the same. Extraction with solvents characterized by different polarity would be more interesting. Second order ANOVA analysis revealed that the Limouni cultivar was the most active extract against all test strains, in other hand Leishmania amazonensis seems to be the most sensitive strain to olive leaf extract with an IC50 < 17.926 ± 1.876 lg/ml (Fig. 4). For L. donovani the IC50 ranged from 2.130 ± 0.023 to 71.570 ± 4.324 lg/ml for the ethanolic extract of Limouni and Zarrazi variety, respectively. The IC50 of the Limouni variety against L. tropica and L. major are respectively, 17.622 and 14.660 lg/ml. Consequently this variety is the stronger inhibitor of these strains. In fact, this activity could be correlated to the amount of phenolic compounds in this cultivar. Compared to the results found by Kyriazis et al. (2013), alcoholic extracts exhibit a stronger activity than aqueous extracts. Indeed, they report that the IC50 of OLWE (olive leaf water extraction) was 60.000 ± 9.5 lg/ml against L. donovani MON-2 promastigotes. Kyriazis et al. (2013) reported that both oleuropein and hydroxytyrosol exhibit a good activity against
Table 3 Leishmanicidal activity of the evaluated olive leaf extracts (IC50 on lg of dry extract/ml). Variety L. amazonensis
L. donovani
L. tropica
L. major
Limouni Ethanol Methanol Mixture Ethanol Methanol Mixture Ethanol Methanol Mixture Ethanol Methanol Mixture
12.353 ± 1.041 6.970 ± 0.316 12.353 ± 0.366 11.234 ± 1.382 2.130 ± 0.023 25.220 ± 3.113 23.614 ± 1.515 17.622 ± 0.651 23.730 ± 1.262 25.613 ± 1.440 14.661 ± 0.380 29.300 ± 2.480
cB aA cB aB aA bC aB bA bB bB aA bC
Zarrazi
Dhokkar
Toffehi
14.379 ± 1.400 cdA 16.950 ± 1.311 dAB 17.926 ± 1.876 dB 17.737 ± 1.430 cA 71.570 ± 4.324 fC 46.533 ± 1.582 dB 46.311 ± 3.228 dB 27.707 ± 2.852 cA 48.942 ± 4.183 dB 54.301 ± 2.260 dB 23.889 ± 1.651 cA 57.293 ± 3.945 dB
4.233 ± 0.023 aB 7.841 ± 0.611 abC 2.936 ± 0.021 aA 16.296 ± 0.63 bcA 26.435 ± 0.843 cB 25.546 ± 1.102 bB 44.011 ± 2.632 dB 19.494 ± 0.785 bA 46.648 ± 1.373 dB 55.734 ± 2.753 dB 23.120 ± 1.581 cA 58.954 ± 0.740 dB
4.017 ± 1.345 8.018 ± 1.657 5.842 ± 0.755 13.785 ± 0.976 56.946 ± 1.265 37.245 ± 1.560 35.612 ± 1.537 17.761 ± 0.860 33.794 ± 2.205 42.159 ± 2.777 17.681 ± 1.383 36.876 ± 1.211
⁄ Mean ± standard deviation. a–b, means within a column row with different letters are significantly different (p < 0.05). A–B, means within a row different letters are significantly different (p < 0.05). NS, non-significant.
Chemlali Tataouine aA abB bAB abA eC cB cB bA cB cC bA cB
16.199 ± 2.020 dC 9.277 ± 1.155 bcB 3.894 ± 0.599 aA 53.8818 ± 3.108 eC 6.201 ± 0.939 bA 14.529 ± 3.250aB 31.653 ± 1.779 bB 18.875 ± 1.253 bA 32.295 ± 2.191 cB 39.506 ± 3.146 cC 17.323 ± 1.066 bA 32.208 ± 1.930 bB
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Fig. 4. Effect of the methanolic extract of Limouni variety on Leishmania amazonensis at 48 h (25 lg/ml) (a), negative control (b).
L. infantum and L. donovani. The relative leishmanicidal activity of the five varieties against L. tropica and L. major, was moderately correlated to phenols content, with a regression coefficient respectively of 0.762 and 0.742 (p < 0.001). The flavonoids content of the different extracts was correlated with the leishmanicidal activity against two L. tropica and major (R2L: Tropica ¼ 0:846 and R2L: Major ¼ 0:777, p < 0.001). Flavonoids are broad classes of plant phenolics that are known to possess a well-established protective effect against membrane lipoperoxidative damages and have long been under investigation for antiparasitic activity (Sen et al., 2004). Mendonça-Filho et al. (2004) have revealed the leishmanicidal activity of polyphenolic-rich extracts from husk fiber of Cocosnucifera Linn. (Palmae). The correlation analysis revealed the absence of correlation between the phenolic compounds especially the flavonoid and the leishmanicidal activity against L. donovani and Leishmania amazonensis, suggesting the presence of other bioactive compounds. The olive leaf are rich in polyphenolic compounds such hydroxytyrosol, tyrosol, benzoic acids, in secoiridoids like the oleuropein, in flavonoids and in triterpenoid such as oleanolic acid, maslinic acid, and ursolic acid (Papoti and Tsimidou, 2009). Several studies, proclaimed the antileishmanial activity of triterpenic acid. Torres-Santos et al. (2004), showed that oleanolic acid and ursolic acid, triterpenic acids isolated from the methanolic fraction of Pourouma guianensis exhibit a strong activity against Leishmania amazonensis. 7. Conclusion Despite the fact that the four Leishmania species tested in this study are able to cause different forms of leishmaniasis, the olive leaf extracts evaluated in this work seems to possess interesting leishmanicidal properties against all of them. Furthermore, this activity could be the result of the presence of several molecules especially phenolic compounds. These results suggest that there could be a potential for new compounds to develop new drugs against leishmaniasis. However, bio guided purification will be necessary to isolate and purified the bioactive compounds from olive leaf extract and further evaluate these observations. Acknowledgments This work was supported by the Grants RICET (project No. RD06/0021/0005 of the programme of Redes Temáticas de Investigación Cooperativa, FIS), Spanish Ministry of Health, Madrid, Spain and the Project FIS PI10/01298 ‘‘Protozoosis emergentes por amebas de vida libre: aislamiento y caracterización molecular, identificación de cepas transportadoras de otros agentes patógenos y búsqueda de quimioterapias efectivas’’ and PI13/00490 ‘‘Protozoosis Emergentes por Amebas de Vida Libre: Aislamiento, Caracterización, Nuevas Aproximaciones Terapéuticas y Traslación
Clínica de los Resultados’’ from the Instituto de Salud Carlos III. I.S. was funded by an alternating Scholarship from the University of Carthage, Tunisian Ministry of Higher Education and Scientific Research. A.L.A. was funded by a Grant ‘‘Ayudas del Programa de Formación de Personal Investigador, para la realización de Tesis Doctorales’’ from the Agencia Canaria de Investigación, Innovación y Sociedad de la Información from the Canary Islands Government. M.R.B. was funded by CEI Canarias, Campus Atlántico Internacional and Becas Fundación Cajacanarias para Postgraduados 2014. C.M.M.N. was supported by a postdoctoral Grant from the Fundación Canaria Manuel Morales, La Palma, Canary Islands. J.L.M. was supported by the Ramón y Cajal Subprogramme from the Spanish Ministry of Science and Innovation RYC-2011-08863. References Andarwulan, N., Batari, R., Sandrasari, D.A., Bolling, B., Wijay, H., 2010. Flavonoid content and antioxidant activity of vegetables from Indonesia. Food Chem. 121, 1231–1235. Aoun, K., Bouratbine, A., Harrat, Z., Belkaid, M., BelHadj Ali, S., 2001. Particular profile of the zymodemes of Leishmania infantum causing visceral leishmaniasis in Tunisia. Bull. Soc. Pathol. Exot. 94, 375–377. Ben Ismail, R., Ben Rachid, R., 1989. Epidémiologie des leishmanioses en Tunisie. In: Maladies Tropicales Transmissibles. AUPELF UREF, John Libbey Eurotex, Paris, pp. 73–80. Bero, J., Hannaert, V., Chataigné, G., Hérent, M.F., Quetin-Leclercq, J., 2011. In vitro antitrypanosomal and antileishmanial activity of plants used in Benin in traditional medicine and bio-guided fractionation of the most active extract. J. Ethnopharmacol. 137, 998–1002. Brand-Williams, W., Cuvelier, M.E., Berset, C., 1995. Use of a free radical method to evaluate antioxidant activity. LWT – Food Sci. Technol. 28, 25–30. Cabrera-Serra, M.G., Lorenzo-Morales, J., Romero, M.L., Valladares, B., Piñero, J.E., 2007. In vitro activity of perifosine: a novel alkylphospholipid against the promastigote stage of Leishmania species. Parasitol. Res. 100, 1155–1157. Camacho, M.D., Mata, R., Castaneda, P., Kirby, G.C., Warhurst, D.C., Croft, S.L., Phillipson, J.D., 2000. Bioactive compounds from Celaenodendron mexicanum. Planta Med. 66, 463–468. Delonrenzi, J.C., Attias, M., Gattas, C.R., Andrade, M., Rezende, C., Pinto, A.D., Henriques, A.T., Bou-Habib, D.C., Saraiva, E.M.B., 2001. Antileishmanial activity of an indole alkaloid from Peschieraaustralis. Antimicrob. Agents Chemother. 45, 1349–1354. Dewanto, V., Wu, X., Adom, K., Liu, R.H., 2002. Thermal processing enhances the nutritional value of tomatoes by increasing total antioxidant activity. J. Agric. Food Chem. 50, 3010–3014. Godinho, J.L., Simas-Rodrigues, C., Silva, R., Ürmenyi, T.P., de Souza, W., Rodrigues, J.C., 2012. Efficacy of miltefosine treatment in Leishmania amazonensis-infected BALB/c mice. Int. J. Antimicrobial. Agents 39, 326–331. Japón-Luján, R., Luque de Castro, M.D., 2006. Superheated liquid extraction of oleuropein and related biophenols from olive leaf. J. Chromatogr. A 1136, 185– 191. Jia, Z.S., Tang, M.C., Wu, J.M., 1999. The determination of flavonoid contents in mulverry and their scavenging effects on superoxide radicals. Food Chem. 64, 555–559. Kayser, O., Kiderlen, A.F., Laatsch, H., Croft, S.L., 2000. In vitro leishmanicidal activity of monomeric and dimeric naphthoquinones. Acta Trop. 77, 307–314. Kyriazis, J.D., Aligiannis, N., Polychronopoulos, P., Skaltsounis, A.L., Dotsika, E., 2013. Leishmanicidal activity assessment of olive tree extracts. Phytomedicine 20, 275–281. Mendonça-Filho, R.R., Rodrigues, I.A., Alviano, D.S., Santos, A.L., Soares, R.M., Alviano, C.S., Lopes, A.H., Rosa, Mdo S., 2004. Leishmanicidal activity of polyphenolic-rich extracts from husk fiber of Cocosnucifera Linn. (Palmae). Res. Microbiol. 155, 136–143.
I. Sifaoui et al. / Experimental Parasitology 141 (2014) 106–111 O’Brien, J., Wilson, I., Orton, T., Pognan, F., 2000. Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur. J. Biochem. 267, 5421–5426. Papoti, V., Tsimidou, M.Z., 2009. Impact of sampling parameters on the radical scavenging potential of olive (Olea europaea L.) leaf. J. Agric. Food Chem. 57, 3470–3477. Pyo, Y.H., Lee, T.C., Logendra, L., Rosen, R.T., 2004. Antioxidant activity and phenolic compounds of Swiss chard. Food Chem. 85, 19–26. Rivero-Pérez, M.D., Muñiz, P., González-Sanjosé, M.L., 2007. Antioxidant profile of red wines evaluated by total antioxidant capacity, scavenger activity and biomarkers of oxidative stress methodologies. J. Agric. Food Chem. 55, 5476–5783. Sauvain, M., Kunesch, N., Poisson, J., Gantier, J.C., Gayral, P., Dedet, J.P., 1996. Isolation of leishmanicidal triterpenes and lignans from the Amazonian liana Doliocarpus dentatus (Dilleniaceae). Phytother. Res. 10, 1–4.
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Sen, G., Mukhopadhaya, R., Ghosal, J., Biswas, T., 2004. Combination of ascorbate and a-tocopherol as a preventive therapy against structural and functional defects of erythrocytes in visceral leishmaniasis. Free Radic. Res. 38, 527– 534. Škerget, M., Kotnik, P., Hadolin, M., Hraš, A.R., Simonicˇ, M., Knez, Zˇ., 2005. Phenols, proanthocyanidins, flavones and flavonols in some plant materials and their antioxidant activities. Food Chem. 89, 191–198. Sun, T., Ho, C.T., 2006. Antioxidant activities of buckwheat extracts. Food Chem. 90, 743–749. Torres-Santos, E.S., Lopes, D., Rodrigues Oliveira, R., Carauta, J.P.P., Bandeira Falcao, C.A., Kaplan, M.A.C., Rossi-Bergmann, B., 2004. Antileishmanial activity of isolated triterpenoids from Pourouma guianensis. Phytomedicine 11, 114–120. World Health Organisation (WHO), 2007. WHO/CDS/NTD/IDM/2007.3. Cutaneous leishmaniasis. Why are you neglecting me?.
