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Contents lists available at ScienceDirect

Experimental Parasitology
journal homepage: www.elsevier.com/locate/yexpr

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3 Bioassay guided isolation and identification of anti-Acanthamoeba

4 compounds from Tunisian olive leaf extracts
7 Q1 Ines Sifaoui a,⇑, Atteneri López-Arencibia b,2, Juan Carlos Ticona c, Carmen Mª Martín-Navarro b,d,2,
8 María Reyes-Batlle b,2, Mondher Mejri a,1, Jacob Lorenzo-Morales b,2, Antonio Ignacio Jiménez c,
9 Basilio Valladares b,2, Isabel Lopez-Bazzocchi c, Manef Abderabba a,1, José E. Piñero b,2
10 a
Laboratoire Matériaux-Molécules et Applications, IPEST, B.P. 51, 2070 La Marsa, University of Carthage, Tunisia
11 b
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
12 c
Instituto Universitario de Bio-Orgánica Antonio González, Universidad de La Laguna, Tenerife, Canary Islands, Spain
13 d
Centre for Integrative Physiology, School of Biomedical Sciences, University of Edinburgh, Edinburgh, Scotland, United Kingdom

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1 1
8
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
319
20  Olive leaf extracts contain an
21 interesting anti-Acanthamoeba
22 activity.
23  The bio-guided fractionation of the
24 extract yielded three known
25 molecules: oleanolic acid, maslinic
26 acid and oleuropein.
27  To the best of our knowledge the
28 activity of the isolated molecules has
29 not been previously reported against
30 amoebae.

33
a r t i c l e i n f o a b s t r a c t
3
4 5
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36 Article history: Pathogenic Acanthamoeba strains are causative agents of Granulomatous Amoebic Encephalitis (GAE) and 48
37 Received 30 November 2013 Acanthamoeba keratitis (AK) worldwide. The existence of the cyst stage complicates Acanthamoeba 49
38 Received in revised form 10 February 2014 therapy as it is highly resistant to antibiotics and physical agents. The aim of this study was to investigate 50
39 Accepted 21 February 2014
the activity of Limouni olive leaf cultivar against the trophozoite stage of Acanthamoeba. The ethyl acetate 51
40 Available online xxxx
and methanol extracts of this variety were tested against Acanthamoeba castellanii Neff. The ethyl 52
acetate extract of olive leaf was the most active showing an IC50 of 5.11 ± 0.71 lg/ml of dry extract. 53
41 Keywords:
Bio-guided fractionation of this extract was conducted and led to the identification of three active com- 54
42 Olive leaf extract
43 Acanthamoeba
pounds namely oleanolic and maslinic acids and oleuropein which could be used for the development of 55
44 Chemotherapy novel therapeutic approaches against Acanthamoeba infections. 56
45 Bioassay fractionation Ó 2014 Elsevier Inc. All rights reserved. 57
46 58

59
60
61 Q2 1. Introduction environments. However, these amoebae can also act as opportunis- 64
tic pathogens causing Granulomatous Amoebic Encephalitis (GAE) 65
62 Acanthamoeba species are ubiquitous free-living amoebae and Acanthamoeba keratitis. The therapy of these diseases has been 66
63 which dwell in several habitats, including air, soil, and water undermined by resistance, variable efficacy between strains or spe- 67
cies, toxicity, and requirement for long courses of treatment. A 68
need for identifying alternative natural and safe sources of 69
⇑ Corresponding author. Fax: +216 71 746 551.
molecules, especially of plant origin, to treat these diseases has 70
E-mail address: ines.sifaoui@hotmail.com (I. Sifaoui).
1
Fax: +216 71 746 551. notably increased in recent years. 71
2
Fax: +34 922318514.

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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72 Among these plants, olive leaves have been used from the past was fractionated by to 14 fractions by chromatography on silica gel 108
73 in traditional medicine to cure many infections such as malaria and (Hexane/AceOH/MeOH). The 14 fractions were concentrated in va- 109
74 ulcers. More recently, pharmaceutical and food industries started cuo. Based on the observed antiprotozoal activity and similarities 110
75 to use several olive products considering their richness in bioactive in TLC profiles, we procedure to combined some fractions. Nine 111
76 molecules. Phytochemical investigations of olive leaves led to the fractions was further recovery. The most active fractions against 112
77 isolation of various secoiridoid and triterpenes, some of which the tested parasite were subjected to silica gel flash chromatogra- 113
78 were found to possess several pharmacological properties (Sifaoui phy or Sephadex gel as illustrated in Fig. 1. 114
79 et al., 2013). Oleuropein and related derivatives constitute the ma-
80 jor class of molecules in olive leaf extracts. Several studies have re- 2.2. In vitro drug sensitivity assay 115
81 ported antioxidant, hypoglycemic, antihypertensive, antimicrobial,
82 antitumoral, antiatherosclerotic, antiparasitic and antiviral, includ- 2.2.1. Amoebic strain 116
83 ing anti-HIV, properties of olive leaves (Lee-Huang et al., 2003; Acanthamoeba castellanii Neff (ATCC 30010), a type strain from 117
84 Somova et al., 2003; Goulas et al., 2009; Sudjana et al., 2009). Ole- the American Type Culture Collection was used in this study. This 118
85 anolic and maslinic acids, a natural penta cyclic triterpene, are strain was axenically grown in PYG medium (0.75% (w/v) proteose 119
86 widely present in dietary plants, especially in olive product. Those peptone, 0.75% (w/v) yeast extract and 1.5% (w/v) glucose) contain- 120
87 compounds have attracted much interest due to their biological ing 40 lg gentamicin ml 1(Biochrom AG, Cultek, Granollers, Barce- 121
88 activities, such as anti-viral (Saija and Uccella, 2000) antidiabeto- lona, Spain) previous it use for the assays. 122
89 genic (Jemai et al., 2009) and anticancer functions (Sánchez-Tena
90 et al., 2013). Our preliminary work with Limouni variety showed
2.2.2. In vitro effect against the trophozoite stage of Acanthamoeba 123
91 that ethyl acetate fraction of it had interesting amoebicidal activ-
The anti-Acanthamoeba activities of the assayed extracts were 124
92 ity. Thus, the objectives of this work were to isolate and identify
determined by the Alamar BlueÒ assay as previously described 125
93 the major molecules responsible for this effect.
(McBride et al., 2005; Martín-Navarro et al., 2008). Briefly, Acantha- 126
moeba strains were seeded in duplicate on a 96-well microtiter 127
94 2. Materials and methods plate with 50 ll from a stock solution of 104 cells ml 1. Amoebae 128
were allowed to adhere for 15 min process which was checked 129
95 2.1. Plant material using a Leika DMIL inverted microscope (Leika, Wetzlar, Germany). 130
After that, 50 ll of serial dilutions of the fraction or the pure com- 131
96 Plant material (leaves) of Limouni variety was harvested from pound was added to each well, (In all tests, 1% dimethyl sulfoxide 132
97 the southeastern part of Tunisia, ‘Ain el Maaguel, Douiret’, during (DMSO; Sigma Chemical Co., St. Louis, Mo.), a concentration that 133
98 the maturing fruit season 2010/2011. The olive leaves collected was used to dissolve the highest dose of the compounds but that 134
99 were ground to a fine powder using a mill. Each shell powder sam- had no effect on the parasite). Finally the Alamar Blue Assay Re- 135
100 ple (0.25 g) was macerated with 20 ml of extraction solvents (first agentÒ (Biosource, Europe, Nivelles, Belgium) was placed into each 136
101 with ethyl acetate than with methanol) in a capped glass tube on well at an amount equal to 10% of the medium volume. Test plates 137
102 an agitating plate at a constant stirring rate (280 rpm) for 1 h containing Alamar Blue were then incubated for 120 h at 28 °C 138
103 and under 55 °C. Afterwards, a rotary vacuum evaporator at 40 °C with a slight agitation. 139
104 was used in order to remove solvent. Subsequently the plates were analyzed, during an interval of 140
time between 72 and 144 h, on a Microplate Reader Model 680 141
105 2.1.1. Bioassay guided fractionation of Limouni ethyl acetate extract (Biorad, Hercules, CA) using a test wavelength of 570 nm and a ref- 142
106 The fractionation of the olive leaf extract was guided by inhib- erence wavelength of 630 nm. Percentages of growth inhibition, 143
107 itory activity against Acanthamoeba Neff. Initially, the crude extract 50% inhibitory concentrations (IC50) were calculated by linear 144

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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145 regression analysis with 95% confidence limits. All experiments 4. Conclusion 192
146 were performed three times each in duplicate, and the mean values
147 were also calculated. Olive leaves showed strong activity potential on the prolifera- 193
tion of A. castellanii Neff trophozoites. These results suggest that 194
148 3. Results and discussion the extract evaluated here is a potential therapeutic drug for the 195
treatment of the parasite infections. The three compounds isolated 196
149 Olives leaf extract, fraction and isolates molecules were screened and identified have been already described in olive leaf extracts 197
150 for their activity against A. castellanii Neff. The IC50/96 h was chosen but their amoebicidal activity had not been investigated previ- 198
151 as the appropriate and comparable data to give as previously ously. However, a further studied to investigate the mode of action 199
152 described (Martín-Navarro et al., 2008; Sifaoui et al., 2013). These of this compound against the parasite tested as well as the synergy 200
153 values are summarized on Table 1. It could be observed that the effects between them. 201
154 amoebicidal activity was based on a dependent-dose application.
155 The parasite have been inhibit by all tested sample with an IC50 ran- Acknowledgments 202
156 ged from 5.96 ± 0.24 lg/ml for the fraction 5 to 25.05 ± 1.1 lg/ml
157 for the fraction number 9. Comparably to other reports, olive extract This work was supported by the Grants RICET (Project No. 203
158 provide a stronger activity against this parasite, indeed, Degerli et al. RD12/0018/0012 of the programme of Redes Temáticas de Investi- 204
159 (2012) report that in the presence of 32 mg/ml of Origanum syria- gación Cooperativa, FIS), Spanish Ministry of Health, Madrid, Spain 205
160 cum and Origanum laevigatum extract, no viable trophozoites were and the Project FIS PI10/01298 ‘‘Protozoosis emergentes porame- 206
161 observed by the third hour. The bioassay guided fractionation of bas de vida libre: aislamiento y caracterización molecular, identifi- 207
162 the crude ethyl acetate extract of Limouni olive leaves yielded three cación de cepas transportadoras de otros agentes patógenos y 208
163 known molecules namely two triterpenic acids (oleanolic and búsqueda de quimioterapias efectivas’’ and PI13/00490 ‘‘Protozoo- 209
164 maslinic acids) and a secoiridoid (oleuropein) identifying by 1H sis Emergentes por Amebas de Vida Libre: Aislamiento, Cara- 210
165 NMR. All those compounds, present a good amoebic activity with cterización, Nuevas Aproximaciones Terapéuticas y Traslación 211
166 an IC50 ranged from 30.88 ± 1.33 to 57.52 ± 0.203 lg/ml respec- Clínica de los Resultados’’ from the Instituto de Salud Carlos III. 212
167 tively for maslinic acid and oleuropein. These compounds have been I.S. was funded by an alternating Scholarship from the Univer- 213
168 already described in olive leaf extracts already described but their sity of Carthage, Tunisian Ministry of Higher Education and Scien- 214
169 amoebicidal activity had not been investigated previously. How- tific Research and by CEI Canarias, Campus Atlántico Internacional. 215
170 ever, several report, have confirmed the therapeutic effect of those A.L.A. was funded by a Grant ‘‘Ayudas del Programa de Formación 216
171 molecules on human and animal cells. Many studies have shown de Personal Investigador, para la realización de Tesis Doctorales’’ 217
172 that oleuropein possesses a wide range of pharmacologic and health from the Agencia Canaria de Investigación, Innovación y Sociedad 218
173 promoting properties including anti-atherogenic (Carluccio et al., de la Información from the Canary Islands Government. C.M.M.N. 219
174 2003), antiviral (Micol et al., 2005), antimicrobial (Saija and Uccella, was supported by a postdoctoral Grant from the Fundación Canaria 220
175 Q3 2000), hypotensive (Khayyal et al., 2002), antidiabetic effects (Jemai Manuel Morales, La Palma, Canary Islands. M.R.B. was funded by 221
176 et al., 2009) and antitumor activity (Elamin et al., 2013). Oleanolic CEI Canarias, Campus Atlántico Internacional and Becas Funda- 222
177 and maslinic acids are a triterpenoid compounds that exist widely ciónCaja canarias para Postgraduados 2014. J.L.M. was supported 223
178 in olive products (Stiti et al., 2007). Their biological effects have by the Ramón y Cajal Subprogramme from the Spanish Ministry 224
179 been demonstrated in many reports such as anti-oxidants (Bala- of Economy and Competivity RYC-2011-08863. 225
180 nehru and Nagarajan, 1991), antifungal, anti-inflammatory, anti-
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301

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