Antileishmanial Potential of Medicinal Plant Extracts from the North-West of Morocco

Accepted Manuscript Antileishmanial potential of medicinal plant extracts from the North-West of Morocco Abdelhakim Bouyahya, Abdeslam Et-Touys, Nadia Dakka, Hajiba Fellah, Jamal Abrini, Youssef Bakri PII: S2314-8535(17)30181-6 DOI: http://dx.doi.org/10.1016/j.bjbas.2017.06.003 Reference: BJBAS 214 To appear in: Beni-Suef University Journal of Basic and Applied Sciences Received Date: 28 April 2017 Revised Date: 1 June 2017 Accepted Date: 12 June 2017 Please cite this article as: A. Bouyahya, A. Et-Touys, N. Dakka, H. Fellah, J. Abrini, Y. Bakri, Antileishmanial potential of medicinal plant extracts from the North-West of Morocco, Beni-Suef University Journal of Basic and Applied Sciences (2017), doi: http://dx.doi.org/10.1016/j.bjbas.2017.06.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. 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Antileishmanial potential of medicinal plant extracts from the North-West of Morocco Abdelhakim Bouyahya1,2*#, Abdeslam Et-Touys1,3#, Nadia Dakka1, Hajiba Fellah3, Jamal Abrini2, Youssef Bakri1 1 Laboratory of Human Pathologies Biology, Department of Biology, Faculty of Sciences, and Genomic Center of Human Pathologies, Mohammed V University, Rabat, Morocco 2 Biology and health Laboratory, Department of Biology, Faculty of Science, Abdelmalek Essaadi University, Tetouan, Morocco 3 National Reference Laboratory of Leishmaniasis, National Institute of Health, Rabat, Morocco Corresponding author: Bouyahya Abdelhakim *E-mail address: [email protected] Postal address : Bouyahya Abdelhakim, Laboratory of Human Pathologies Biology, Faculty of Sciences of Rabat, University Mohammed V of Rabat 4, Av. Ibn battouta BP1014 Rabat- Morocco. # Both authors contribute equally to the carrying out of the study Antileishmanial potential of medicinal plant extracts from the North-West of Morocco Abstract The aim of this study is to evaluate the antileishmanial activity of selected medicinal plants from the North-West of Morocco. Plant extracts were prepared by maceration using methanol, ethanol, and n-hexane. The antileishmanial activity was evaluated against Leishmania major, Leishmania tropica, and Leishmania infantum using MTT (3- (4.5-dimethylthiazol- 2yl)-2.5- diphenyltetrazolium bromide) assay. All plant extracts showed a reducing in cell promastigotes viability with variability depending on tested strains and type of extracts. The n-hexane extract showed the highest antileishmanial activity and L. infantum was the most sensitive parasite. The best growth inhibition was observed with Cistus crispus n-hexane extract against L. major (IC50=47.29±2.25 µg/mL), Arbutus unedo n-hexane extract against L. infantum (IC50=64.05±1.44 µg/mL) and Arbutus unedo n-hexane extract against L. tropica (IC50=79.57±2.66 µg/mL). Considering these results, medicinal plants from the North-West of Morocco could constitute a promoter source for antileishmanial compounds. Keywords: Leishmaniasis; medicinal plants; antileishmanial activity; Ouezzane 1. Introduction Leishmaniasis is considered as a serious health problem worldwide, especially in Africa where it significant morbidity and mortality (Chiheb et al., 1999; El Aasri et al., 2014). It is caused by parasites transmitted by phlebotomy insects (Bates, 2007; Dostalova & Volf, 2012). Recently, the number of researches on antileishmanial agents significantly increased for two reasons: firstly, the seed effects demonstrated by several treatments such as antimony derivatives which remain toxic and expensive. Secondly, several Leishmania species showed the resistance against synthetic molecules, and therefore the emergence and reemergence of infectious diseases. These two situations have oriented pharmacological researches on antileishmanial drugs to screened molecules that possess a selective efficacy and tolerable safety. Medicinal plant secondary metabolites showed several pharmacological properties such as antibacterial (Bouyahya et al., 2017a ; Bouyahya et al., 2017b), antioxidant (Bouyahya et al., 2017b; Bouyahya et al., 2016a), antitumor (Bouyahya et al., 2016b ; Aneb et al., 2016), antifungal (Fadel et al., 2013), anti-litholitic (Khouchlaa et al., 2017a ; Khouchlaa et al., 2017b) and antileishmanial activities (Et-Touys et al., 2016a ; Et-Touys et al., 2016b). These secondary metabolites are complex molecules with various functional structures such as polyphenols, flavonoids, terpenoids and coumarins (Lahlou, 2013). In this way, recent studies that focused on antileishmanial activities of medicinal plant products showed the success of these products in the inhibition of growth of several Leishmania species such as L. major (cutaneous leishmaniasis) and L. infantum (visceral leishmaniasis) (Et-Touys et al., 2016a ; Et-Touys et al., 2016b ; Essid et al., 2015). The North-West of Morocco (province of Ouezzane) is rich in medicinal plants and some of them are endemic (Ennabili et al., 2000; Merzouki et al., 2000). However, there is an under exploitation of the explored species. We have demonstrated in early published studies that some selected medicinal plants from this region have important charges of phenolic and flavonoid contents and possess significant antibacterial and antioxidant effects (Bouyahya et al., 2016b; Bouyahya et al., 2017b). In this context, the aim of this study was the screening of the antileishmanial activity of some selected plant extracts against Leishmania species. 2. Materials and methods 2.1. Collection of plant material and preparation of organic extracts Medicinal plants were collected from the North-West of Morocco (Ouezzane province: Zoumi area). The scientific name, family, vernacular name, part plant collected, medicinal use and pharmacological properties of these plants are given in table 1. The collected parts were dried in the dark at room temperature and then they were ground to obtain the powder. This later was extracted by maceration using methanol, ethanol, and n-hexane. After 72 hours of maceration, the plant extracts were filtered and the solvent was eliminated using a rotary evaporator. 2.2. Total phenolic and flavonoid contents The total phenolic contents (TPC) and the total flavonoid contents were determined in our previous studies (Bouyahya et al., 2016b; Bouyahya et al., 2016c; Bouyahya et al., 2017c; Bouyahya et al., 2017d). The TPC was estimated by the Folin Ciocalteu assay using gallic acid as standard. The TFC was estimated by the aluminum chloride (AlCl3) colorimetric assay using quercetin as standard. 2.3. Evaluation of the antileishmanial Activity 2.3.1. Culture of leishmania species The Leishmania species tested in this study are: Leishmania infantum (MHOM/MA/1998/LVTA), Leishmania tropica (MHOM/MA/2010/LCTIOK-4), and Leishmania major (MHOM/MA/2009/LCER19-09). These three species were isolated and identified from Moroccan infected patients at the National Reference Laboratory of Leishmaniasis, National Institute of Health, Rabat-Morocco. The species were cultivated as described by Et-Touys et al. (2016b). Briefly, parasite cultures of each Leishmania species were washed with phosphate-buffered saline (PBS) and centrifuged at 1500 rpm for 10 minutes. Cells were then re-suspended in RPMI 1640 (GIBCO) supplemented with 10% of heat-inactivated fetal calf serum and 1% of Penicillin-Streptomycin mixture. Cultures were maintained at 23°C. 2.3.2. Antileishmanial activity Before evaluating the antileishmanial activity, the cellular density of each species was calculated using light microscopy. When cellular density s was reached a threshold 6 concentration of 10 cells/mL, L. infantum, L. tropica, and L. major promastigotes were washed twice with phosphate-buffered saline (PBS) and centrifuged at 2500 rpm for 10 minutes. To evaluate the anti-promastigote activity, 100 µL of parasites culture were resuspended in a 96-well tissue culture plate, in fresh culture medium according to Et-Touys et al. (2016b). Briefly, parasites were incubated at 2.5x106 cells/well for 72 hours at 23◦C in the presence of various concentrations of extract (µg/mL) dissolved in 1% DMSO. DMSO was used at a final concentration never exceeding 1% which is not toxic to parasites (Essid et al., 2015; Oliveira et al., 2011). Sterile PBS and 1% DMSO (vehicle) were used as negative controls, while Glucantime® was used as positive control. 2.3.3. Cell viability assay The viability of Leishmania species was evaluated using the MTT (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) colorimetric assay as described by Essid et al. (2015). Briefly, 10 µL of MTT (10 mg/mL) were added to each micro-well and incubated for 3 hours at 30◦ C. The reaction was stopped by addition of 100 L of 50% (v/v) isopropanol-10% (w/v) sodium dodecyl sulfate (SDS) mixture to each well in order to dissolve insoluble formazan formed after tetrazolium dye reduction. After 30 minutes of incubation at room temperature, absorbance was measured at 560 nm using an ELISA plate reader. All assays were conducted in triplicate and compared to negative control (parasites) and reference drug (Glucantime). Cell viability was also evaluated by determination of the extract concentrations which inhibited half of the cell

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