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Contents lists available at ScienceDirect
Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr 5 6
Bioassay guided isolation and identification of anti-Acanthamoeba compounds from Tunisian olive leaf extracts
3 4 7 8 9 10 11 12 13
Q1
Ines Sifaoui a,⇑, Atteneri López-Arencibia b,2, Juan Carlos Ticona c, Carmen Mª Martín-Navarro b,d,2, María Reyes-Batlle b,2, Mondher Mejri a,1, Jacob Lorenzo-Morales b,2, Antonio Ignacio Jiménez c, Basilio Valladares b,2, Isabel Lopez-Bazzocchi c, Manef Abderabba a,1, José E. Piñero b,2 a
Laboratoire Matériaux-Molécules et Applications, IPEST, B.P. 51, 2070 La Marsa, University of Carthage, 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 Instituto Universitario de Bio-Orgánica Antonio González, Universidad de La Laguna, Tenerife, Canary Islands, Spain d Centre for Integrative Physiology, School of Biomedical Sciences, University of Edinburgh, Edinburgh, Scotland, United Kingdom b c
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h i g h l i g h t s
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Olive leaf extracts contain an
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interesting anti-Acanthamoeba activity. The bio-guided fractionation of the extract yielded three known molecules: oleanolic acid, maslinic acid and oleuropein. To the best of our knowledge the activity of the isolated molecules has not been previously reported against amoebae.
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Article history: Received 30 November 2013 Received in revised form 10 February 2014 Accepted 21 February 2014 Available online xxxx Keywords: Olive leaf extract Acanthamoeba Chemotherapy Bioassay fractionation
a b s t r a c t Pathogenic Acanthamoeba strains are causative agents of Granulomatous Amoebic Encephalitis (GAE) and Acanthamoeba keratitis (AK) worldwide. The existence of the cyst stage complicates Acanthamoeba therapy as it is highly resistant to antibiotics and physical agents. The aim of this study was to investigate the activity of Limouni olive leaf cultivar against the trophozoite stage of Acanthamoeba. The ethyl acetate and methanol extracts of this variety were tested against Acanthamoeba castellanii Neff. The ethyl acetate extract of olive leaf was the most active showing an IC50 of 5.11 ± 0.71 lg/ml of dry extract. Bio-guided fractionation of this extract was conducted and led to the identification of three active compounds namely oleanolic and maslinic acids and oleuropein which could be used for the development of novel therapeutic approaches against Acanthamoeba infections. Ó 2014 Elsevier Inc. All rights reserved.
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Q2 1. Introduction Acanthamoeba species are ubiquitous free-living amoebae which dwell in several habitats, including air, soil, and water ⇑ Corresponding author. Fax: +216 71 746 551. 1 2
E-mail address: [email protected] (I. Sifaoui). Fax: +216 71 746 551. Fax: +34 922318514.
environments. However, these amoebae can also act as opportunistic pathogens causing Granulomatous Amoebic Encephalitis (GAE) and Acanthamoeba keratitis. The therapy of these diseases has been undermined by resistance, variable efficacy between strains or species, toxicity, and requirement for long courses of treatment. A need for identifying alternative natural and safe sources of molecules, especially of plant origin, to treat these diseases has notably increased in recent years.
http://dx.doi.org/10.1016/j.exppara.2014.02.018 0014-4894/Ó 2014 Elsevier Inc. All rights reserved.
Please cite this article in press as: Sifaoui, I., et al. Bioassay guided isolation and identification of anti-Acanthamoeba compounds from Tunisian olive leaf extracts. Exp. Parasitol. (2014), http://dx.doi.org/10.1016/j.exppara.2014.02.018
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Among these plants, olive leaves have been used from the past in traditional medicine to cure many infections such as malaria and ulcers. More recently, pharmaceutical and food industries started to use several olive products considering their richness in bioactive molecules. Phytochemical investigations of olive leaves led to the isolation of various secoiridoid and triterpenes, some of which were found to possess several pharmacological properties (Sifaoui et al., 2013). Oleuropein and related derivatives constitute the major class of molecules in olive leaf extracts. Several studies have reported antioxidant, hypoglycemic, antihypertensive, antimicrobial, antitumoral, antiatherosclerotic, antiparasitic and antiviral, including anti-HIV, properties of olive leaves (Lee-Huang et al., 2003; Somova et al., 2003; Goulas et al., 2009; Sudjana et al., 2009). Oleanolic and maslinic acids, a natural penta cyclic triterpene, are widely present in dietary plants, especially in olive product. Those compounds have attracted much interest due to their biological activities, such as anti-viral (Saija and Uccella, 2000) antidiabetogenic (Jemai et al., 2009) and anticancer functions (Sánchez-Tena et al., 2013). Our preliminary work with Limouni variety showed that ethyl acetate fraction of it had interesting amoebicidal activity. Thus, the objectives of this work were to isolate and identify the major molecules responsible for this effect.
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2. Materials and methods
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2.1. Plant material
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Plant material (leaves) of Limouni variety was harvested from the southeastern part of Tunisia, ‘Ain el Maaguel, Douiret’, during the maturing fruit season 2010/2011. The olive leaves collected were ground to a fine powder using a mill. Each shell powder sample (0.25 g) was macerated with 20 ml of extraction solvents (first with ethyl acetate than with methanol) 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.
72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92
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2.1.1. Bioassay guided fractionation of Limouni ethyl acetate extract The fractionation of the olive leaf extract was guided by inhibitory activity against Acanthamoeba Neff. Initially, the crude extract
was fractionated by to 14 fractions by chromatography on silica gel (Hexane/AceOH/MeOH). The 14 fractions were concentrated in vacuo. Based on the observed antiprotozoal activity and similarities in TLC profiles, we procedure to combined some fractions. Nine fractions was further recovery. The most active fractions against the tested parasite were subjected to silica gel flash chromatography or Sephadex gel as illustrated in Fig. 1.
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2.2. In vitro drug sensitivity assay
115
2.2.1. Amoebic strain Acanthamoeba castellanii Neff (ATCC 30010), a type strain from the American Type Culture Collection was used in this study. This strain was axenically grown in PYG medium (0.75% (w/v) proteose peptone, 0.75% (w/v) yeast extract and 1.5% (w/v) glucose) containing 40 lg gentamicin ml 1(Biochrom AG, Cultek, Granollers, Barcelona, Spain) previous it use for the assays.
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2.2.2. In vitro effect against the trophozoite stage of Acanthamoeba The anti-Acanthamoeba activities of the assayed extracts were determined by the Alamar BlueÒ assay as previously described (McBride et al., 2005; Martín-Navarro et al., 2008). Briefly, Acanthamoeba strains were seeded in duplicate on a 96-well microtiter plate with 50 ll from a stock solution of 104 cells ml 1. Amoebae were allowed to adhere for 15 min process which was checked using a Leika DMIL inverted microscope (Leika, Wetzlar, Germany). After that, 50 ll of serial dilutions of the fraction or the pure compound was added to each well, (In all tests, 1% dimethyl sulfoxide (DMSO; Sigma Chemical Co., St. Louis, Mo.), a concentration that was used to dissolve the highest dose of the compounds but that had no effect on the parasite). Finally the Alamar Blue Assay ReagentÒ (Biosource, Europe, Nivelles, Belgium) was placed into each well at an amount equal to 10% of the medium volume. Test plates containing Alamar Blue were then incubated for 120 h at 28 °C with a slight agitation. Subsequently the plates were analyzed, during an interval of time between 72 and 144 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 growth inhibition, 50% inhibitory concentrations (IC50) were calculated by linear
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Fig. 1. Bioassay guided isolation of anti-Acanthamoeba compounds olive leaf extracts.
Please cite this article in press as: Sifaoui, I., et al. Bioassay guided isolation and identification of anti-Acanthamoeba compounds from Tunisian olive leaf extracts. Exp. Parasitol. (2014), http://dx.doi.org/10.1016/j.exppara.2014.02.018
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regression analysis with 95% confidence limits. All experiments were performed three times each in duplicate, and the mean values were also calculated.
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3. Results and discussion
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Olives leaf extract, fraction and isolates molecules were screened for their activity against A. castellanii Neff. The IC50/96 h was chosen as the appropriate and comparable data to give as previously described (Martín-Navarro et al., 2008; Sifaoui et al., 2013). These values are summarized on Table 1. It could be observed that the amoebicidal activity was based on a dependent-dose application. The parasite have been inhibit by all tested sample with an IC50 ranged from 5.96 ± 0.24 lg/ml for the fraction 5 to 25.05 ± 1.1 lg/ml for the fraction number 9. Comparably to other reports, olive extract provide a stronger activity against this parasite, indeed, Degerli et al. (2012) report that in the presence of 32 mg/ml of Origanum syriacum and Origanum laevigatum extract, no viable trophozoites were observed by the third hour. The bioassay guided fractionation of the crude ethyl acetate extract of Limouni olive leaves yielded three known molecules namely two triterpenic acids (oleanolic and maslinic acids) and a secoiridoid (oleuropein) identifying by 1H NMR. All those compounds, present a good amoebic activity with an IC50 ranged from 30.88 ± 1.33 to 57.52 ± 0.203 lg/ml respectively for maslinic acid and oleuropein. These compounds have been already described in olive leaf extracts already described but their amoebicidal activity had not been investigated previously. However, several report, have confirmed the therapeutic effect of those molecules on human and animal cells. Many studies have shown that oleuropein possesses a wide range of pharmacologic and health promoting properties including anti-atherogenic (Carluccio et al., 2003), antiviral (Micol et al., 2005), antimicrobial (Saija and Uccella, Q3 2000), hypotensive (Khayyal et al., 2002), antidiabetic effects (Jemai et al., 2009) and antitumor activity (Elamin et al., 2013). Oleanolic and maslinic acids are a triterpenoid compounds that exist widely in olive products (Stiti et al., 2007). Their biological effects have been demonstrated in many reports such as anti-oxidants (Balanehru and Nagarajan, 1991), antifungal, anti-inflammatory, antihyperlipidemia, hepatoprotective, antiproliferative (Martín-Navarro et al., 2008), immunomodulatory, (Price et al., 1987; Nishino et al., 1988), anti-HIV (Kashiwada et al., 2000), anti-arrhythmic and cardiotonic (Somova et al., 2004). Although, it could be observed that the original crude extract (ethyl acetate extract) exhibit a stronger amoebicidal activity than the pure compound, suggesting the presence of synergetic activities between components. Several reports have confirmed that the fractionation of biologically-active crude extracts can lead to the loss of their original activity, due to the synergistically and additively effects among the present components (Ibikunle et al., 2011; Misbah et al., 2013).
Table 1 Anti-amoebic activity of the olive leaf fractions. Drug substance
IC50 (lg/ml) at 96 h
Ethyl acetate extract Methanolic extract Fraction 1 Fraction 2 Fraction 3 Fraction 4 Fraction 5 Fraction 6 Fraction 7 Fraction 8 Fraction 9 Oleanolic acid Maslinic acid Oleuropein
5.11 ± 0.71 9.54 ± 0.33 >100 19.19 ± 0.65 6.56 ± 0.23 12.5 ± 0.98 17.39 ± 0.88 5.96 ± 0.24 12.5 ± 0.45 12.01 ± 1.2 25.05 ± 1.1 43.67 ± 3.34 30.88 ± 1.33 57.52 ± 0.203
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4. Conclusion
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Olive leaves showed strong activity potential on the proliferation of A. castellanii Neff trophozoites. These results suggest that the extract evaluated here is a potential therapeutic drug for the treatment of the parasite infections. The three compounds isolated and identified have been already described in olive leaf extracts but their amoebicidal activity had not been investigated previously. However, a further studied to investigate the mode of action of this compound against the parasite tested as well as the synergy effects between them.
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Acknowledgments
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This work was supported by the Grants RICET (Project No. RD12/0018/0012 of the programme of Redes Temáticas de Investigación Cooperativa, FIS), Spanish Ministry of Health, Madrid, Spain and the Project FIS PI10/01298 ‘‘Protozoosis emergentes poramebas 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 and by CEI Canarias, Campus Atlántico Internacional. 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. C.M.M.N. was supported by a postdoctoral Grant from the Fundación Canaria Manuel Morales, La Palma, Canary Islands. M.R.B. was funded by CEI Canarias, Campus Atlántico Internacional and Becas FundaciónCaja canarias para Postgraduados 2014. J.L.M. was supported by the Ramón y Cajal Subprogramme from the Spanish Ministry of Economy and Competivity RYC-2011-08863.
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References
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Balanehru, S., Nagarajan, B., 1991. Protective effect of oleanolic acid and ursolic acid against lipid peroxidation. Biochem. Int. 24 (5), 981–990. Carluccio, M.A., Siculella, L., Ancora, M.A., Massaro, M., Scoditti, E., Storelli, C., Visioli, F., Distante, A., De Caterina, R., 2003. Olive oil and red wine antioxidant polyphenols inhibit endothelial activation: antiatherogenic properties of Mediterranean diet phytochemicals. Arterioscler. Thromb. Vasc. Biol. 23 (4), 622–629. Degerli, S., Tepe, B., Celiksoz, A., Berk, S., Malatyali, E., 2012. In vitro amoebicidal activity of Origanum syriacum and Origanum laevigatum on Acanthamoeba castellanii cysts and trophozoites. Exp. Parasitol. 131 (1), 20–24. Elamin, M.H., Daghestani, M.H., Omer, S.A., Elobeid, M.A., Virk, P., Al-Olayan, E.M., Hassan, Z.K., Mohammed, O.B., Aboussekhra, A., 2013. Olive oil oleuropein has anti-breast cancer properties with higher efficiency on ER-negative cells. Food Chem. Toxicol. 53, 310–316. Goulas, V., Exarchou, V., Troganis, A.N., Psomiadou, E., Fotsis, T., Briasoulis, E., Gerothanassis, I.P., 2009. Phytochemicals in olive-leaf extracts and their antiproliferative activity against cancer and endothelial cells. Mol. Nutr. Food Res. 53 (5), 600–608. Ibikunle, G.F., Adebajo, A.C., Famuyiwa, F.G., Aladesanmi, A.J., Adewunmi, C.O., 2011. In-vitro evaluation of anti-trichomonal activities of eugenia uniflora leaf. Afr. J. Tradit. Complement. Altern. Med. 8 (2), 170–176. Jemai, H., El Feki, A., Sayadi, S., 2009. Antidiabetic and antioxidant effects of hydroxytyrosol and oleuropein from olive leaves in alloxan-diabetic rats. J. Agric. Food. Kashiwada, Y., Nagao, T., Hashimoto, A., Ikeshiro, Y., Okabe, H., Cosentino, L.M., Lee, K.H., 2000. Anti-AIDS agents 38. Anti-HIV activity of 3-O-acyl ursolic acid derivatives. J. Nat. Prod. 63 (12), 1619–1622. Lee-Huang, S., Zhang, L., Huang, P., Chang, Y., Huang, P., 2003. Anti-HIV activity of olive leaf extract (OLE) and modulation of host cell gene expression by HIV-1 infection and OLE treatment. Biochem. Biophys. Res. Commun. 307 (4), 1029– 1037. Martín-Navarro, C.M., Lorenzo-Morales, J., Cabrera-Serra, M.G., Rancel, F., CoronadoAlvarez, N.M., Piñero, J.E., Valladares, B., 2008. The potential pathogenicity of chlorhexidine-sensitive Acanthamoeba strains isolated from contact lens cases
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from asymptomatic individuals in Tenerife, Canary Islands, Spain. J. Med. Microbiol. 57 (1), 1399–1404. McBride, J., Ingram, P.R., Henríquez, F.L., 2005. Development of colorimetric microtiter plate assay for assessment of antimicrobials against Acanthamoeba. J. Clin. Microbiol. 43 (2), 629–634. Micol, V., Caturla, N., Perez-Fons, L., Mas, V., Perez, L., Estepa, A., 2005. The olive leaf extract exhibits antiviral activity against viral haemorrhagic septicaemia rhabdovirus (VHSV). Antiviral Res. 66 (2–3), 129–136. Misbah, H., Aziz, A.A., Aminudin, N., 2013. Antidiabetic and antioxidant properties of Ficus deltoidea fruit extracts and fractions. BMC Complement. Altern. Med. 13, 118. Nishino, H., Nishino, A., Takayasu, J., Hasegawa, T., Iwashima, A., Hirabayashi, K., Iwata, S., Shibata, S., 1988. Inhibition of the tumor-promoting action of 12-otetradecanoylphorbol-13-acetate by some oleanane-type triterpenoid compounds. Cancer Res. 48 (18), 5210–5215. Price, K.R., Johnson, L.T., Fenwick, G.R., 1987. The chemistry and biological significance of saponins in foods and feedingstuffs. Crit. Rev. Food Sci. Nutr. 26 (1), 27–135. Saija, A., Uccella, N., 2000. Olive biophenols: functional effects on human wellbeing. Trends Food Sci. Technol. 11 (9–10), 357–363.
Sánchez-Tena, S., Reyes-Zurita, F.J., Díaz-Moralli, S., Vinardell, M.P., Reed, M., GarcíaGarcía, F., Dopazo, J., Lupiáñez, J.A., Günther, U., Cascante, M., 2013. Maslinic acid-enriched diet decreases intestinal tumorigenesis in Apc (Min/+) mice through transcriptomic and metabolomic reprogramming. PLoS One 8 (3), e59392. Sifaoui, I., López-Arencibia, A., Martín-Navarro, C.M., Chammem, N., Mejri, M., Lorenzo-Morales, J., Abderabba, M., Piñero, J.E., 2013. Activity assessment of Tunisian olive leaf extracts against the trophozoite stage of Acanthamoeba. Parasitol. Res. 112 (8), 2825–2829. Somova, L.I., Shode, F.O., Mipando, M., 2004. Cardiotonic and antidysrhythmic effects of oleanolic acid and ursolic acids, methyl maslinate and uvaol. Phytomedicine 11 (2–3), 121–129. Somova, L.I., Shode, F.O., Ramnanan, P., Nadar, A., 2003. Antihypertensive, antiatherosclerotic and antioxidant activity of triterpenoids isolated from Olea europaea, subspecies Africana leaves. J. Ethnopharmacol. 84 (2–3), 299–305. Stiti, N., Triki, T.S., Hartmann, M.A., 2007. Formation of triterpenoids throughout Olea europaea fruit ontogeny. Lipids 42 (1), 55–67. Sudjana, A.N., D’Orazio, C., Ryan, V., Rasool, N., Ng, J., Islam, N., Riley, T.V., Hammer, K.A., 2009. Antimicrobial activity of commercial Olea europaea (olive) leaf extract. Int. J. Antimicrob. Agents 33 (5), 461–463.
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No. of Pages 4, Model 5G
15 April 2014 Experimental Parasitology xxx (2014) xxx–xxx 1
Contents lists available at ScienceDirect
Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr 5 6
Bioassay guided isolation and identification of anti-Acanthamoeba compounds from Tunisian olive leaf extracts
3 4 7 8 9 10 11 12 13
Q1
Ines Sifaoui a,⇑, Atteneri López-Arencibia b,2, Juan Carlos Ticona c, Carmen Mª Martín-Navarro b,d,2, María Reyes-Batlle b,2, Mondher Mejri a,1, Jacob Lorenzo-Morales b,2, Antonio Ignacio Jiménez c, Basilio Valladares b,2, Isabel Lopez-Bazzocchi c, Manef Abderabba a,1, José E. Piñero b,2 a
Laboratoire Matériaux-Molécules et Applications, IPEST, B.P. 51, 2070 La Marsa, University of Carthage, 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 Instituto Universitario de Bio-Orgánica Antonio González, Universidad de La Laguna, Tenerife, Canary Islands, Spain d Centre for Integrative Physiology, School of Biomedical Sciences, University of Edinburgh, Edinburgh, Scotland, United Kingdom b c
15 14 16 1 1 8 3 19
h i g h l i g h t s
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Olive leaf extracts contain an
21 22
interesting anti-Acanthamoeba activity. The bio-guided fractionation of the extract yielded three known molecules: oleanolic acid, maslinic acid and oleuropein. To the best of our knowledge the activity of the isolated molecules has not been previously reported against amoebae.
23 24 25 26 27 28 29 30
g r a p h i c a l a b s t r a c t
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a r t i c l e 3 4 5 7 36 37 38 39 40 41 42 43 44 45 46
i n f o
Article history: Received 30 November 2013 Received in revised form 10 February 2014 Accepted 21 February 2014 Available online xxxx Keywords: Olive leaf extract Acanthamoeba Chemotherapy Bioassay fractionation
a b s t r a c t Pathogenic Acanthamoeba strains are causative agents of Granulomatous Amoebic Encephalitis (GAE) and Acanthamoeba keratitis (AK) worldwide. The existence of the cyst stage complicates Acanthamoeba therapy as it is highly resistant to antibiotics and physical agents. The aim of this study was to investigate the activity of Limouni olive leaf cultivar against the trophozoite stage of Acanthamoeba. The ethyl acetate and methanol extracts of this variety were tested against Acanthamoeba castellanii Neff. The ethyl acetate extract of olive leaf was the most active showing an IC50 of 5.11 ± 0.71 lg/ml of dry extract. Bio-guided fractionation of this extract was conducted and led to the identification of three active compounds namely oleanolic and maslinic acids and oleuropein which could be used for the development of novel therapeutic approaches against Acanthamoeba infections. Ó 2014 Elsevier Inc. All rights reserved.
48 49 50 51 52 53 54 55 56 57 58 59
60 61 62 63
Q2 1. Introduction Acanthamoeba species are ubiquitous free-living amoebae which dwell in several habitats, including air, soil, and water ⇑ Corresponding author. Fax: +216 71 746 551. 1 2
E-mail address: [email protected] (I. Sifaoui). Fax: +216 71 746 551. Fax: +34 922318514.
environments. However, these amoebae can also act as opportunistic pathogens causing Granulomatous Amoebic Encephalitis (GAE) and Acanthamoeba keratitis. The therapy of these diseases has been undermined by resistance, variable efficacy between strains or species, toxicity, and requirement for long courses of treatment. A need for identifying alternative natural and safe sources of molecules, especially of plant origin, to treat these diseases has notably increased in recent years.
http://dx.doi.org/10.1016/j.exppara.2014.02.018 0014-4894/Ó 2014 Elsevier Inc. All rights reserved.
Please cite this article in press as: Sifaoui, I., et al. Bioassay guided isolation and identification of anti-Acanthamoeba compounds from Tunisian olive leaf extracts. Exp. Parasitol. (2014), http://dx.doi.org/10.1016/j.exppara.2014.02.018
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Among these plants, olive leaves have been used from the past in traditional medicine to cure many infections such as malaria and ulcers. More recently, pharmaceutical and food industries started to use several olive products considering their richness in bioactive molecules. Phytochemical investigations of olive leaves led to the isolation of various secoiridoid and triterpenes, some of which were found to possess several pharmacological properties (Sifaoui et al., 2013). Oleuropein and related derivatives constitute the major class of molecules in olive leaf extracts. Several studies have reported antioxidant, hypoglycemic, antihypertensive, antimicrobial, antitumoral, antiatherosclerotic, antiparasitic and antiviral, including anti-HIV, properties of olive leaves (Lee-Huang et al., 2003; Somova et al., 2003; Goulas et al., 2009; Sudjana et al., 2009). Oleanolic and maslinic acids, a natural penta cyclic triterpene, are widely present in dietary plants, especially in olive product. Those compounds have attracted much interest due to their biological activities, such as anti-viral (Saija and Uccella, 2000) antidiabetogenic (Jemai et al., 2009) and anticancer functions (Sánchez-Tena et al., 2013). Our preliminary work with Limouni variety showed that ethyl acetate fraction of it had interesting amoebicidal activity. Thus, the objectives of this work were to isolate and identify the major molecules responsible for this effect.
94
2. Materials and methods
95
2.1. Plant material
96
Plant material (leaves) of Limouni variety was harvested from the southeastern part of Tunisia, ‘Ain el Maaguel, Douiret’, during the maturing fruit season 2010/2011. The olive leaves collected were ground to a fine powder using a mill. Each shell powder sample (0.25 g) was macerated with 20 ml of extraction solvents (first with ethyl acetate than with methanol) 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.
72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92
97 98 99 100 101 102 103 104
105 106 107
2.1.1. Bioassay guided fractionation of Limouni ethyl acetate extract The fractionation of the olive leaf extract was guided by inhibitory activity against Acanthamoeba Neff. Initially, the crude extract
was fractionated by to 14 fractions by chromatography on silica gel (Hexane/AceOH/MeOH). The 14 fractions were concentrated in vacuo. Based on the observed antiprotozoal activity and similarities in TLC profiles, we procedure to combined some fractions. Nine fractions was further recovery. The most active fractions against the tested parasite were subjected to silica gel flash chromatography or Sephadex gel as illustrated in Fig. 1.
108
2.2. In vitro drug sensitivity assay
115
2.2.1. Amoebic strain Acanthamoeba castellanii Neff (ATCC 30010), a type strain from the American Type Culture Collection was used in this study. This strain was axenically grown in PYG medium (0.75% (w/v) proteose peptone, 0.75% (w/v) yeast extract and 1.5% (w/v) glucose) containing 40 lg gentamicin ml 1(Biochrom AG, Cultek, Granollers, Barcelona, Spain) previous it use for the assays.
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2.2.2. In vitro effect against the trophozoite stage of Acanthamoeba The anti-Acanthamoeba activities of the assayed extracts were determined by the Alamar BlueÒ assay as previously described (McBride et al., 2005; Martín-Navarro et al., 2008). Briefly, Acanthamoeba strains were seeded in duplicate on a 96-well microtiter plate with 50 ll from a stock solution of 104 cells ml 1. Amoebae were allowed to adhere for 15 min process which was checked using a Leika DMIL inverted microscope (Leika, Wetzlar, Germany). After that, 50 ll of serial dilutions of the fraction or the pure compound was added to each well, (In all tests, 1% dimethyl sulfoxide (DMSO; Sigma Chemical Co., St. Louis, Mo.), a concentration that was used to dissolve the highest dose of the compounds but that had no effect on the parasite). Finally the Alamar Blue Assay ReagentÒ (Biosource, Europe, Nivelles, Belgium) was placed into each well at an amount equal to 10% of the medium volume. Test plates containing Alamar Blue were then incubated for 120 h at 28 °C with a slight agitation. Subsequently the plates were analyzed, during an interval of time between 72 and 144 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 growth inhibition, 50% inhibitory concentrations (IC50) were calculated by linear
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Fig. 1. Bioassay guided isolation of anti-Acanthamoeba compounds olive leaf extracts.
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regression analysis with 95% confidence limits. All experiments were performed three times each in duplicate, and the mean values were also calculated.
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3. Results and discussion
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Olives leaf extract, fraction and isolates molecules were screened for their activity against A. castellanii Neff. The IC50/96 h was chosen as the appropriate and comparable data to give as previously described (Martín-Navarro et al., 2008; Sifaoui et al., 2013). These values are summarized on Table 1. It could be observed that the amoebicidal activity was based on a dependent-dose application. The parasite have been inhibit by all tested sample with an IC50 ranged from 5.96 ± 0.24 lg/ml for the fraction 5 to 25.05 ± 1.1 lg/ml for the fraction number 9. Comparably to other reports, olive extract provide a stronger activity against this parasite, indeed, Degerli et al. (2012) report that in the presence of 32 mg/ml of Origanum syriacum and Origanum laevigatum extract, no viable trophozoites were observed by the third hour. The bioassay guided fractionation of the crude ethyl acetate extract of Limouni olive leaves yielded three known molecules namely two triterpenic acids (oleanolic and maslinic acids) and a secoiridoid (oleuropein) identifying by 1H NMR. All those compounds, present a good amoebic activity with an IC50 ranged from 30.88 ± 1.33 to 57.52 ± 0.203 lg/ml respectively for maslinic acid and oleuropein. These compounds have been already described in olive leaf extracts already described but their amoebicidal activity had not been investigated previously. However, several report, have confirmed the therapeutic effect of those molecules on human and animal cells. Many studies have shown that oleuropein possesses a wide range of pharmacologic and health promoting properties including anti-atherogenic (Carluccio et al., 2003), antiviral (Micol et al., 2005), antimicrobial (Saija and Uccella, Q3 2000), hypotensive (Khayyal et al., 2002), antidiabetic effects (Jemai et al., 2009) and antitumor activity (Elamin et al., 2013). Oleanolic and maslinic acids are a triterpenoid compounds that exist widely in olive products (Stiti et al., 2007). Their biological effects have been demonstrated in many reports such as anti-oxidants (Balanehru and Nagarajan, 1991), antifungal, anti-inflammatory, antihyperlipidemia, hepatoprotective, antiproliferative (Martín-Navarro et al., 2008), immunomodulatory, (Price et al., 1987; Nishino et al., 1988), anti-HIV (Kashiwada et al., 2000), anti-arrhythmic and cardiotonic (Somova et al., 2004). Although, it could be observed that the original crude extract (ethyl acetate extract) exhibit a stronger amoebicidal activity than the pure compound, suggesting the presence of synergetic activities between components. Several reports have confirmed that the fractionation of biologically-active crude extracts can lead to the loss of their original activity, due to the synergistically and additively effects among the present components (Ibikunle et al., 2011; Misbah et al., 2013).
Table 1 Anti-amoebic activity of the olive leaf fractions. Drug substance
IC50 (lg/ml) at 96 h
Ethyl acetate extract Methanolic extract Fraction 1 Fraction 2 Fraction 3 Fraction 4 Fraction 5 Fraction 6 Fraction 7 Fraction 8 Fraction 9 Oleanolic acid Maslinic acid Oleuropein
5.11 ± 0.71 9.54 ± 0.33 >100 19.19 ± 0.65 6.56 ± 0.23 12.5 ± 0.98 17.39 ± 0.88 5.96 ± 0.24 12.5 ± 0.45 12.01 ± 1.2 25.05 ± 1.1 43.67 ± 3.34 30.88 ± 1.33 57.52 ± 0.203
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4. Conclusion
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Olive leaves showed strong activity potential on the proliferation of A. castellanii Neff trophozoites. These results suggest that the extract evaluated here is a potential therapeutic drug for the treatment of the parasite infections. The three compounds isolated and identified have been already described in olive leaf extracts but their amoebicidal activity had not been investigated previously. However, a further studied to investigate the mode of action of this compound against the parasite tested as well as the synergy effects between them.
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Acknowledgments
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This work was supported by the Grants RICET (Project No. RD12/0018/0012 of the programme of Redes Temáticas de Investigación Cooperativa, FIS), Spanish Ministry of Health, Madrid, Spain and the Project FIS PI10/01298 ‘‘Protozoosis emergentes poramebas 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 and by CEI Canarias, Campus Atlántico Internacional. 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. C.M.M.N. was supported by a postdoctoral Grant from the Fundación Canaria Manuel Morales, La Palma, Canary Islands. M.R.B. was funded by CEI Canarias, Campus Atlántico Internacional and Becas FundaciónCaja canarias para Postgraduados 2014. J.L.M. was supported by the Ramón y Cajal Subprogramme from the Spanish Ministry of Economy and Competivity RYC-2011-08863.
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