Sesquiterpenoids and flavonoids from Inula viscosa induce programmed cell death in kinetoplastids

Contents lists available at ScienceDirect Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha Original article Sesquiterpenoids and flavonoids from Inula viscosa induce programmed cell death in kinetoplastids Ikrame Zeouk a,b,c, *, Ines Sifaoui a,b,d , Atteneri López-Arencibia a,b,d , María Reyes-Batlle a,b,d , Carlos J. Bethencourt-Estrella a,b,d , Isabel L. Bazzocchi e , Khadija Bekhti c , Jacob Lorenzo-Morales a,b,d , Ignacio A. Jiménez e , José E. Piñero a,b,d a Instituto Universitario de Enfermedades Tropicales y Salud Pública de Canarias, Universidad de La Laguna, Avda. Astrofísico Fco. Sánchez, S/N, La Laguna, Tenerife, Islas Canarias 38203, Spain b Red de Investigación Colaborativa en Enfermedades Tropicales (RICET) c Departement of Biology, Sidi Mohamed Ben Abdellah University, Faculty of Sciences and Techniques, Laboratory of Microbial Biotechnology and Bioactive Molecules, PB 2202, Fez, Morocco d Departamento de Obstetricia, Ginecología, Pediatría, Medicina Preventiva y Salud Pública, Toxicología, Medicina Legal y Forense y Parasitología, Universidad De La Laguna, La Laguna, Tenerife, Islas Canarias 38203, Spain e Instituto Universitario de Bio-Orgánica Antonio González, Departamento de Química Orgánica, Universidad de La Laguna, Avenida Astrofísico Francisco Sánchez 2, 38206 La Laguna, Tenerife, Spain ARTICLE INFO Keywords: Inula viscosa Leishmanicidal Trypanocidal Sesquiterpenoids Flavonoids Programmed cell death ABSTRACT Neglected tropical diseases such as leishmaniasis and American trypanosomiasis represent an increasing health problem. Current treatments are not satisfactory which remains an urgent need for novel, cheap and safe chemotherapies. In the course of our ongoing search for new potential anti-protozoal agents, this study aimed to perform a bio-guided fractionation of Inula viscosa (Asteraceae) using in vitro assays against three strains of Leishmania and Trypanosma genus. Eight known compounds were identified from the ethanolic extract of leaves, sesquiterpenoids (3and 4) and flavonoids (5and 6) were characterized as the main bioactive constituents. Sesquiterpene lactones 3and 4(IC 50 values between 4.99 and 14.26 μM) showed promising antiparasitic activity against promastigotes of L. donovani,L. amazonensis and epimastigotes of T. cruzi. Their structures were successfully characterized by spectroscopic techniques including 1D and 2D NMR experiments. Furthermore, the main bioactive compounds 4,5and 6displayed higher potency (IC 50 values between 0.64 and 2.13 μM) against amastigotes of L. amazonensis than miltefosine (IC 50 3.11 μM), and a low toxicity on macrophages cell line (SI > 45). The analysis of structure-activity relationship (SAR) of the anti-protozoal activity revealed that lactonization or oxidation enhanced the biological profile, suggesting that the hydrophobic moiety was presumably involved in the activity by increasing the affinity and/or cell membrane permeability. In order to get an insight into the mechanism of action of these compounds, programmed cell death (PCD) experiments were performed, and the obtained results suggest that the reported compounds induced PCD in the treated parasites. These results highlight that sesquiterpenoids and flavonoids from I. viscosa could constitute an interesting scaffold for the development of novel antikinetoplastid agents. 1. Introduction Parasitic diseases caused by kinetoplastids such as Leishmania and Trypanosoma species, known as neglected tropical diseases (NTDs), are responsible for high mortality, disability and morbidity rates [1]. According to World Health Organization (WHO), American trypanosomiasis, also known as Chagas disease, is a potentially life-threatening illness caused by the protozoan parasite Trypanosoma cruzi with approximately more than 10 000 deaths per year, and more than 25 million people are at a higher risk of acquiring this disease [2]. Furthermore, leishmaniasis is a complex infectious disease with a varied spectrum of clinical manifestations, which range from self-healing cutaneous ulceration to progressive and lethal visceral infection [3]. It is prevalent in more than 98 endemic countries in the world with an https://doi.org/10.1016/j.biopha.2020.110518 Received 9 May 2020; Received in revised form 24 June 2020; Accepted 2 July 2020 ⁎ Corresponding author at: Instituto Universitario de Enfermedades Tropicales y Salud Pública de Canarias, Universidad de La Laguna, Avda. Astrofísico Fco. Sánchez, S/N, La Laguna, Tenerife, Islas Canarias 38203, Spain. E-mail address: [email protected] (I. Zeouk). Biomedicine & Pharmacotherapy 130 (2020) 110518 Available online 13 July 2020 0753-3322/ © 2020 The Authors. Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). T estimated annual incidence of almost 0.2 to 0.4 million new cases of visceral manifestation and 0.7–1.2 million new cases of cutaneous form [4]. The emergence of resistance, toxicity and high cost of the current treatment reveal the urgent need of alternative chemotherapeutic agents. In this context, more interest has been given to the development of new efficient drugs using science advances such as nanotechnology [5], chemical synthesis [6] and natural products [7]. Among these approaches, plants present a rich source of bioactive compounds [8]. For instance, Asteraceae family has mainly presented promising antiparasitic activities against Leishmania and Trypanosoma genus [9]. Belonging to this family, Inula viscosa also known as Dittrichia viscosa is a medicinal perennial herb native to the Mediterranean basin and has been widely used in traditional medicine to treat different diseases [10].Previous chemical investigations have characterized various phytochemicals in I. viscosa such as flavonoids, sesquiterpenes lactones, acids and glycolipids with a wide range of therapeutic indications [11–13]. Indeed, flavonoids and sesquiterpene lactones are a group of natural products widely described as potent antiparasitic agents [14,15]. Recently, quercetin related compounds have targeted the arginase in Leishmania amazonensis showing a potent activity [16], and artemisinin which is an illustrative example for sesquiterpene lactones, has displayed antiparasitic potency [17] through the induction of apoptotic-like cell death [18,19]. Indeed, cell death of different developmental stages of kinetoplastids from Trypanosoma and Leishmania genus have been coupled to the occurrence of apoptotic markers such as DNA fragmentation, cell shrinkage, chromatin condensation, loss of mitochondrial membrane potential ΔΨ and membrane blebbing [20–22]. Hence, in the present work we report a bio-guided fractionation of I. viscosa ethanolic extract in order to characterize leishmanicidal and trypanosomal compounds, to elucidate their mechanism of action targeting the PCD, and to analyze the structure-activity relationship (SAR) involved in their anti-protozoal activity. 2. Materials and methods 2.1. General procedure, chemicals and reagents Optical rotations were determined on a Perkin-Elmer 241 automatic polarimeter. NMR spectra, ROESY (spin lock field 2500 Hz), HSQC and HMBC (optimized for J =7.7 Hz) were performed on a Bruker Avance 500 and 600 spectrometers at 300°K. Silica gel 60 (particle size 15–40 and 63–200 μm, Macherey-Nagel) was used for column chromatography, while silica gel 60 F254 (Macherey-Nagel) was used for analytical or preparative TLC and Sephadex LH-20 (Sigma-Aldrich) were used for the column chromatographic (CC) separation. Centrifugal preparative TLC was performed using a Chromatotron (Harrison Research Inc. model 7924 T) on 4or 1-mm silica gel 60 PF254 disks with flow rate 2−4 mL min −1 . The spots were visualized by UV light and heating silica gel plates sprayed with H 2 OH 2 SO 4 -AcOH (1:4:20). All the used solvents were analytical grade from Panreac. Reagents, deuterated solvents were purchased from Sigma-Aldrich, benznidazole from Aldrich and miltefosine from Æterna Zentaris. For biological tests, Schneider’s medium (Sigma-Aldrich), RPMI 1640 and LIT media (Gibco®), alamarBlue®reagent (Invitrogen, Life Technologies), EnSpire®Multimode Plate Reader (Perkin Elmer), and Leika DMIL inverted microscope (Leika, Wetzlar, Germany) were used. 2.2. Plant material identification and extraction In the present work, the studied plants have been selected from an ethnopharmacological study undertaken in the central north of Morocco. The used parts of the most recommended plants as described by herbalists have been collected in the Atlas Mountains, Imouzzer region-Morocco in July 2017, then identified by botanists. In order to obtain the crude extracts, cleaned used parts of the different plants were shade dried at room temperature and subsequently milled with a simple commercial electric grinder. Powdered material was extracted by maceration in ethanol (1:10 w/v) for 6 h at room temperature with continuous stirring at 500 rpm (revolutions per minute). The resulting mixture was filtered using Whatman filter n°1 and the solvent was concentrated under vacuum in a rotary evaporator at 45 °C, dried extracts were stored in a refrigerator at 4 °C until further use. Amongst the sixteen tested plants, I. viscosa extract (5 g, 5%) was the most active against the three evaluated parasites; therefore, it was selected for further bioassays. The geographical coordinates for this species are 33°55′37 N, 5°2′50 W and a voucher specimen (RAB107342) has been deposited in the Herbarium of the Scientific Institute of Rabat, Morocco. 2.3. Bio-guided fractionation procedure The prepared EtOH extracts were assayed for their antiprotozoal activity against promastigotes of L. amazonensis and L. donovani and epimastigotes of T. cruzi. After that, a bio-guided fractionation methodology was used to identify antiprotozoal compounds in the ethanolic extract of I. viscosa as the most active plant. Thus, the active EtOH extract (5 g) was subjected to CC on silica gel eluted with mixtures of hexane-EtOAc (100:0 to 0:100, 0.5 L each one) of increasing polarity to afford twelve fractions that were combined into seven fractions (F1-F7) based on their TLC profile. The antiprotozoal activity was focused on the active fractions F2 (670.6 mg), F3 (871.5 mg) and F4 (1.7155 g), which were subjected to column chromatography on sephadex LH-20 column, using a MeOH−CHCl 3 system eluent (1:1, 2 L) to afford between forty and fifty six sub-fractions which were combined again based on their TLC profiles (F2/1 to F2/5), (F3/1 to F3/6) and (F4/1 to F4/12), respectively. Indeed, the sub-fractions F3/1 (638.84 mg) and F3/2 (103.56 mg) showed a promising activity against the three tested strains and were subjected to multiple chromatographic steps on silica gel, involving medium-pressure liquid chromatography, centrifugal planar chromatography and preparative TLC using mixtures of hexaneEtOAc, hexane-Et 2 O, CH 2 Cl 2 -EtOAc and CH 2 Cl 2 -acetone as eluent to yield compounds 3(1.78 mg), 4(38.5 mg), 5(3.6 mg) and 7(1.95 mg). Following the same purification procedures, fraction F2 yielded compounds 1(3.3 mg) and 3, while fraction F4 afforded compounds 2 (8.5 mg), 6(28.5 mg) and 8(5.4 mg). 2.4. Leishmanicidal and trypanocidal assays 2.4.1. Parasite strains The antiparasitic activity of crude extract, fractions and pure compounds of I. viscosa was evaluated against the promastigote stage of Leishmania donovani (MHOM/IN/90/GE1F8R), promastigote and amastigote forms of Leishmania amazonensis (MHOM/BR/77/LTB0016) and epimastigote form of Trypanosoma cruzi (Y strain). 2.4.2. In vitro antileishmanial evaluation 2.4.2.1. Anti-promastigotes assay. The bioassay was performed using the alamarBlue®method as previously described [23]. Promastigotes of L. donovani and L. amazonensis were grown at 26 °C in RPMI 1640 modified medium (Gibco) and supplemented with 10 % heatinactivated foetal bovine serum. Cultures in the logarithmic phase were seeded in sterilized 96-well microtiter plates (Corning™) (10 6 parasites/mL) containing the samples dissolved in dimethyl sulfoxide 1% (DMSO) at the suitable concentration to be tested in serial dilutions using Leishmania medium (RPMI 1640) to get a final volume of 200 μL per well, then 20 μL of alamarBlue®were added to the entire plate. After an incubation of 72 h, the plate was checked up visually using an inverted microscope, then analyzed by an EnSpire multimode plate reader (PerkinElmer, MA, USA) using a test wavelength of 570 nm and a reference wavelength of 630 nm. Leishmanicidal activity was expressed as IC 50 values (the concentration of a sample which caused I. Zeouk, et al. Biomedicine & Pharmacotherapy 130 (2020) 110518 2 a 50 % reduction in parasite viability). Those values were calculated by linear regression analysis with 95 % confidence limits. 2.4.2.2. Anti-amastigotes assay. The active pure compounds from I. viscosa were tested against the intra-macrophagic stage of L. amazonensis as previously described in literature [24]. In a 96-well flat bottom plate, the J774A.1 cell line macrophages were cultured at a density of 2 × 10 5 cells/mL in RPMI 1640 medium supplemented with 10 % heat-inactivated foetal bovine serum and incubated for one hour at 37 °C in a 5 % CO 2 atmosphere in order to allow almost complete attachment of the cells. After that, macrophages were infected with L. amazonensis promastigotes in the stationary phase (7 days old culture) with a ratio of 1:10 (macrophage/ parasite) at a concentration of 2×10 6 cells/mL, then incubated at 37 °C in 5% CO 2 for 24 h. Wells were washed with medium to remove non-phagocytosed promastigotes. Thereafter, the infected macrophages were treated with the pure compounds (dissolved in DMSO at the desired concentration) for 24 h. The medium was removed carefully to be replaced by 30 μLof 0.05 % SDS and the plate was shaken for 30 s. Subsequently, 170 μLof Schneider’s medium were added to each well to give a final volume of 200 μL and 20 μL of alamarBlue®were added to the plate and incubated at 26 °C for 72 h in order to give enough time to the transformation from promastigotes to amastigotes. After 72 h of incubation, plates were analyzed using the same protocol of promastigotes test. 2.4.3. In vitro evaluation on T. cruzi epimastigotes The extract, fractions and pure molecules were tested against the epimastigotes of T. cruzi. Briefly, in 96-well plates samples dissolved in DMSO were serially diluted in 100 μL of LIT medium supplemented with 10 % heat-inactivated fetal bovine serum to obtain the desire concentrations selected from the first screening. In all tests, 1 % DMSO was used to dissolve the highest dose of the compounds without inducing any effects on the parasites. After that, epimastigotes in logarithmic growth phase were counted, adjusted to 5 × 10 5 cells/mL, distributed in the previous 96-well plate and incubated at 27 °C for 72 h. The plate was observed under an inverted microscope after 72 h of incubation and analyzed statistically as described in the leishmanicidal test. 2.5. Assessment of cytotoxicity on macrophages The cytotoxicity assay of the pure active compounds was performed as described in literature [25]. Briefly, 2 × 10 5 of macrophages culture were placed in a 96-well plate for 2 h in a 5 % CO 2 incubator at 37 °C, then serial dilutions of the 4 active molecules were prepared in deep well, then transmitted to the plate containing the culture of macrophages giving a final volume of 100 μL. Finally, the plate was incubated in 5 % CO 2 atmosphere for 24 h. The percentage of cell viability was evaluated using the alamarBlue®assay (10 %). Dose response curves were plotted and the CC 50 values were calculated. 2.6. Mechanisms of cell death In order to analyze the programmed cell death pathway induced in the studied parasites, a common step between the four kits was performed. Briefly, parasites were treated with the tested compounds at their IC 90 for 24 h. After that, cells were centrifuged (1500 rpm for 10 min), washed twice with PBS (phosphate buffered saline) and incubated with the suitable reagent of each marker as described below. For the four assays, an untreated control was used. After incubations, an EVOS FL Cell Imaging System AMF4300, Life Technologies, Madrid, Spain was used to observe and analyze the obtained results in order to score and to count cells. 2.6.1. Chromatin condensation determination To analyze the compacted state of chromatin in apoptotic cells, a double-stain apoptosis detection kit (Hoechst 33342/PI) (Life Technologies) was used as recommended by the manufacturer. The strains were incubated with Hoechst 33342 at 5 μg/mL and PI at 1 μg/ mL. After 15 min of incubation at 26 °C, samples were analyzed. 2.6.2. Analysis of mitochondrial membrane potential The JC-1 Mitochondrial Membrane Potential Assay Kit (Cayman Chemical) was used to measure the collapse of an electrochemical gradient across the mitochondrial membrane. Treated promastigotes resuspended in JC-1 buffer were incubated with JC-1 reagent (1:10 v/v) and then incubated at 26 °C for 30 min. Analysis for mean green and red fluorescence intensity and depolarization of the mitochondrial membrane potential was analyzed. 2.6.3. Determination of ATP level ATP level was measured using a Cell Titer-Glo®Luminescent Cell Viability Assay (Promega). The effect of the compounds on the ATP production was evaluated by incubating parasitic strains (10 6 cells/mL) with the tested molecules at their IC 90 for 24 h. 2.6.4. Plasma membrane permeability To detect the plasma membrane permeability of parasites, the SYTOX®Green assay was performed. The treated parasites were incubated with SYTOX®Green at a final concentration of 1 μM (Molecular Probes) for 15 min in the dark at room temperature. The increase in fluorescence due to binding of the fluorescent marker to the parasitic DNA was observed. 2.6.5. Oxidative stress CellRox Deep Red Oxidative Stress Reagent (Thermo Fisher Scientific) was used to measure the generation of Reactive Oxygen Species (ROS) in cells exhibiting strong fluorogenic signal under oxidative state. The assay involves the incubation of the treated parasites with 5 μM of CellRox Reagent for 30 min at 26 °C, then the centrifugation of cells to be re-suspended in buffer. H 2 O 2 at 600 μM for 30 min was used as positive control. The signal for Deep Red is localized in the cytoplasm. Table 1 Leishmanicidal and trypanosomal activity against promastigotes of L. amazonensis and L. donovani and epimastigotes of T. cruzi of the extract, fractions and sub-fractions from leaves of I. viscosa. Samples L. amazonensis IC 50 (μg/mL) L. donovani IC 50 (μg/mL) T. cruzi IC 50 (μg/mL) Crude extract 18.10 ± 2.08 10.87 ± 2.11 12.18 ± 0.50 F2 35.66 ± 1.78 31.07 ± 2.17 8.67 ± 0.45 F2/4 15.95 ± 0.80 52.95 ± 3.18 13.74 ± 0.96 F3 3.80 ± 0.23 4.48 ± 0.15 6.61 ± 0.87 F3/1 6.44 ± 0.02 7.71 ± 2.46 9.04 ± 1.86 F3/2 12.72 ± 1.27 10.66 ± 2.35 23.93 ± 1.91 F4 3.63 ± 0.14 27.12 ± 01.27 27.00 ± 0.87 F4/3 15.46 ± 0.75 12.52 ± 0.42 11.18 ± 1.13 F4/4 17.85 ± 0.85 6.6 ± 1.22 10.76 ± 2.12 F4/5 12.77 ± 0.05 9.08 ± 0.66 8.59 ± 1.58 F4/6 19.60 ± 2.65 18.01 ± 0.90 9.89 ± 0.08 F4/7−9 13.03 ± 1.11 10.76 ± 2.90 5.41 ± 1.38 F4/10 32.17 ± 1.93 36.62 ± 1.46 22.13 ± 1.90 F4/10/1 7.64 ± 0.86 7.93 ± 0.15 2.20 ± 0.49 Miltefosine 2.64 ± 0.10 1.35 ± 0.11 – Benznidazole –– 1.81 ± 0.50 IC 50 : Inhibitory Concentration that inhibits 50 % of the growth of the tested parasite. IC 50 : Means ±Standard deviation. Fractions and sub-fractions that showed IC 50 > 100 μg/mL were excluded. I. Zeouk, et al. Biomedicine & Pharmacotherapy 130 (2020) 110518 3 2.7. Statistical analysis All assays were carried out in triplicate. The results were defined as the mean values of three experiments. The obtained inhibition curves was performed using the Sigma Plot 12.0 software program (Systat Software Inc.). Statistical analyses were performed using the GraphPad Prism 8.0.2. Fluorescence intensity (RFU) was performed using ImageJ software program as the means values of three repetitions. Differences between the values were assessed using a one-way analysis of variance (ANOVA). Data are presented as means ± SD and p< 0.05 was considered statistically significant. Fig. 1. Flowchart of antiprotozoal bio-guided fractionation of Inula viscosa leaves against promastigotes of L. amazonensis and L. donovani and epimastigotes of T. cruzi (Tc). IC 50 : Inhibitory concentration that inhibits 50 % of the growth of the tested parasite. IC 50 values in μg/mL. Fig. 2. Chemical structure of natural compounds 1-8isolated from leaves of Inula viscosa. I. Zeouk, et al. Biomedicine & Pharmacotherapy 130 (2020) 110518 4 3. Results and discussion 3.1. Antikinetoplastid activity Research on antikinetoplastid drugs has been intensified in recent years exploiting natural products from medicinal plants [26,27]. The leishmanicidal and trypanosomal activities of plant extracts have been attributed to several compounds belonging to different chemical groups such as monoterpenes, triterpenes, alkaloids, lignans and flavonoids [28–30]. In special, sesquiterpenoids and flavonoids isolated from various plants have widely exhibited a prominent leishmanicidal [31] and trypanocidal activity [32]. In the present work, ethanolic extracts of sixteen plants used in traditional medicine for treatment of infectious diseases such as skin disorders, in the central north of Morocco, were evaluated. Roots of Alkanna tintoria,Berberis hispanica,Ephedra altissima and Rubia tinctorium; aerial parts of Crataegus oxyacantha and Urtica dioica; leaves of Ammi majus, Globularia alypum,Inula viscosa,Lavandula dentata,Nerium oleander,Origanum majorana, and Rhamnus alaternus; seeds of Eruca sativa and Juniperus oxycedrus and peel of Punica granatum. The extracts were firstly screened at 400, 200, 100; 50 and 25 μg/ mL against promastigote stage of L. donovani and L. amazonensis, and epimastigote form of T. cruzi. The crude ethanolic extract of I. viscosa showed the most interesting activity against the tested strains, with IC 50 s between 10.87 and 18.10 μg/mL (Table 1) which was a promising fact to continue with the bioassay-guided fractionation in order to isolate and identify the main active molecules involved in the antiparasitic effects. Therefore, the ethanolic extract was submitted to liquid chromatography on silica gel affording six fractions, F1-F6 (Fig. 1). Indeed, samples that led to a percentage of growth inhibition (%GI) > 50 % were assayed in different concentrations to determine the half maximal inhibitory concentration (IC 50 ) and were classified as highly active (IC 50 <25μg/mL), active (25 < IC 50 <50μg/mL), moderately active (50 < IC 50 < 100 μg/mL) and not active when IC 50 > 100 μg/mL. In addition, miltefosine and benznidazole were evaluated for comparative purposes as reference drugs against leishmaniasis and Chagas disease respectively. Miltefosine showed IC 50 sof 2.64 μg/mL and 1.35 μg/mL against L. amazonensis and L. donovani, respectively, whereas benznidazole showed an IC 50 of 1.81 μg/mL against T. cruzi. The IC 50 values varied upon the fractions and the parasitic strains showing different magnitudes of the inhibitory potential. The most active fractions, F2 (IC 50 values ranging from 8.67 to 35.66 μg/mL), F3 (IC 50 values ranging from 3.80 to 6.61 μg/mL) and F4 (IC 50 values ranging from 3.63 to 27.12 μg/mL), exhibited potent activity against the three studied parasites, similar to the reference drugs (Table 1 and Fig. 1), highlighting these three fractions as the most promising ones. Subsequently, these fractions were further chromatographed on a sephadex LH-20 column yielding five (F2/1-F2/5), six (F3/1-F3/6) and ten sub-fractions (F4/1-F4/10), respectively, which were assayed against the three parasites. From Fraction 2, F2/4 exhibited a potent antikinetoplastid effect on L. amazonensis (IC 50 15.95 μg/mL) and T. cruzi (IC 50 13.74 μg/mL). From fraction F3, sub-fractions F3/1 and F3/2 were the most actives against the three parasites, showing IC 50 s between 6.44 and 12.72 μg/mL for L. amazonensis and L. donovani, respectively, and an IC 50 of 9.04 μg/mL on T. cruzi for F3/1.While from fraction F4, sub-fractions F4/3, F4/4, F4/5 and F4/6 were the most actives against the three parasites, showing IC 50 s between 6.60 and 19.60 μg/mL for L. amazonensis and L. donovani, respectively and IC 50 s between 8.59 and 11.18 μg/mL for T. cruzi. Therefore, sub-fraction F2/ 4 was submitted to purification steps, affording the sesquiterpenoids 1 and 3. Additionally, sub-fraction F3/1 showed to be the most potent one, yielding the compound 3and sub-fraction F3/2 afforded Table 2 Leishmanicidal, trypanocidal, cytotoxic activity and selectivity index against promastigotes of Leismania spp., epimastigotes of T. cruzi and murine macrophages of the active sesquiterpenoids and flavonoids isolated from I. viscosa. Compounds L. amazonensis L. donovani T. cruzi Murine macrophages IC 50 (μM) SI IC 50 (μM) SI IC 50 (μM) SI CC 50 (μM) 3 9.53 ± 2.44 1.92 11.06 ± 2.33 1.66 4.99 ± 0.04 3.67 18.44 ± 1.30 4 12.12 ± 0.64 3.50 14.26 ± 1.69 2.97 12.52 ± 0.16 3.38 42.36 ± 2.54 5 81.08 ± 3.24 1.18 86.45 ± 2.60 1.11 36.17 ± 2.17 2.65 95.93 ± 0.50 6 44.86 ± 2.95 > 7.73 54.46 ± 2.36 > 6.3 82.95 ± 1.65 > 4.18 > 100 Miltefosine 6.48 ± 0.24 11.14 3.31 ± 0.27 21.79 ––72.18 ± 1.25 Benznidazole ––––6.95 ± 1.92 57.51 399.91 ± 1.04 IC 50 : Inhibitory Concentration that inhibits 50 % of the growth of the tested parasite. IC 50 : Means ±Standard deviation. CC 50 : Cytotoxic Concentration that reduces 50 % of the murine macrophages’viability. SI: Selectivity Index (CC 50 /IC 50 ). Compounds that showed IC 50 > 100 μM were excluded. Table 3 Leishmanicidal activity and selectivity index of select compounds against amastigote stage of L. amazonensis. Compounds L. amazonensis IC 50 (μM) SI a 3 6.98 ± 0.42 2.63 4 0.64 ± 0.08 65.75 5 2.13 ± 1.21 45.00 6 1.91 ± 0.83 > 181.81 Miltefosine 3.11 ± 0.29 23.16 IC 50 : Inhibitory Concentration that inhibits 50 % of the growth of the tested parasite. IC 50 : Means ±Standard deviation. CC 50 : Cytotoxic Concentration that reduces 50 % of the murine macrophages’ viability. SI a : Selectivity Index (amastigotes) (CC 50 /IC 50 ). Table 4 Leishmanicidal and trypanocidal activity of the selected compounds for study of mechanism of action. Compounds IC 90 (μM) L. donovani L. amazonensis T. cruzi 3 76.25 ± 3.05 12.85 ± 1.07 17.00 ± 0.33 4 94.23 ± 0.57 48.53 ± 0.18 22.11 ± 0.30 5 107.66 ± 1.76 322.42 ± 0.97 191.68 ± 0.77 6 135.57 ± 0.20 158.23 ± 3.37 173.45 ± 0.61 Miltefosine 5.15 ± 0.50 9.63 ± 0.50 – Benznidazole ––25.591 ± 2.728 IC 90 :Effective Concentration that inhibits 90 % of the growth of the tested parasite. IC 90 : Means ±Standard deviation. I. Zeouk, et al. Biomedicine & Pharmacotherapy 130 (2020) 110518 5 compounds 4,5and 7. Sub-fraction F4/10 yielded compounds 2,6and 8(Figs. 1,2). Hence, in some cases, the IC 50 was higher than the crude extract which could be explained by a possible synergism between I. viscosa components since the complex mixtures of plant components may provide different outcomes: an additive, a synergistic or an antagonistic effect. The chemical structures of the known isolated Fig. 3. Leishmania amazonensis promastigotes incubated with IC 90 of the four active compounds for 24 h: Inuloxin A (B,G,L), sakuranetin (C,H,M), 8-epi-xanthatin1β,5β-epoxide (D,I,N), taxifolin (E,J,O). Images (40X) are representative of the cell population observed in the performed experiments using an EVOS FL Cell Imaging System AMF4300, Life Technologies, USA. Hoechst stain is different when comparing negative control (A,F,K) with treated cells where the nuclei are bright blue. Red fluorescence corresponds to the propidium iodide stain. Hoechst stain (F-J), Propidium iodide stain (K-O). The bar graph includes the calculated values of RFU (Fluorescence intensity). Differences between the values were assessed using one-way analysis of variance (ANOVA). Data are presented as means ± SD (N = 3) and letters a-e or A*-E* reflect that means within compounds with different letters are significantly different (p< 0.05). (For interpretation of the references to colour in the Figure, the reader is referred to the web version of this article). Fig. 4. Images (40X) obtained from an EVOS FL Cell Imaging System showing fluorescence when promastigotes of Leishmania donovani incubated with IC 90 of the four tested compounds for 24 h: Inuloxin A (B,G,L), sakuranetin (C,H,M), 8-epi-xanthatin-1β,5β-epoxide (D,I,N), taxifolin (E,J,O) and stained with Hoechstpropidium iodide. Hoechst corresponds to the chromatin condensation (blue) in treated cells. Red fluorescence corresponds to the propidium iodide stain. A control without treatment has been used (A,F,K). The bar graph includes the calculated values of RFU (Fluorescence intensity). Differences between the values were assessed using one-way analysis of variance (ANOVA). Data are presented as means ± SD (N = 3) and letters a-e or A*-E* reflect that means within compounds with different letters are significantly different (p< 0.05). (For interpretation of the references to colour in the Figure, the reader is referred to the web version of this article). Fig. 5. Images (40X) obtained from EVOS FL Cell Imaging System showing the effect of Inuloxin A (B,G,L), sakuranetin (C,H,M), 8-epi-xanthatin-1β,5β-epoxide (D, I,N), taxifolin (E,J,O) at the IC 90 against epimastigotes of Trypanosoma cruzi after 24 h of incubation in comparison to the control (A,F,K), then using Hoechstpropidium iodide double stains. Hoechst corresponds to the chromatin condensation (blue) in treated cells. Red fluorescence corresponds to the propidium iodide stain. The bar graph includes the calculated values of RFU (Fluorescence intensity). Differences between the values were assessed using one-way analysis of variance (ANOVA). Data are presented as means ± SD (N = 3) and letters a-e or A*-E* reflect that means within compounds with different letters are significantly different (p< 0.05). (For interpretation of the references to colour in the Figure, the reader is referred to the web version of this article). I. Zeouk, et al. Biomedicine & Pharmacotherapy 130 (2020) 110518 6 compounds (1-8) were identified as isocostic acid (1)[33], ilicic acid (2)[34], 8-epi-xanthatin-1β,5β-epoxide (3)[35], inuloxin A (4)[36], sakuranetin (5)[37], taxifolin (6)[38], 3-O-acetyl-7-O-methylaromadendrin (7)[38], and quercetin (8)[39] using spectrometric and spectroscopic data, including 1D and 2D NMR experiments, in addition to comparison with data reported in the literature. Compounds 1-8 were evaluated against the three parasites. In addition, the cytotoxicity on murine macrophages was also assessed. Miltefosine showed a CC 50 of 72.18 μM, whereas benznidazole showed a CC 50 of 399.91 μM (Table 2). The results, over the promastigote stage of Leishmania, showed that compounds 3and 4displayed potent activity in μM range and were slightly less potent than the reference drug. Moreover, compounds 3and 4showed similar potency to benznidazole against epimastigotes of T. cruzi. Regarding the selectivity index (SI) towards macrophages (Table 2), the compounds 3and 4showed a moderated cytotoxic profile on the three parasites (SI values ranging from 1.66 to 3.67). Based on the in vitro results on promastigote forms, compounds 3-6were selected to be evaluated on intracellular amastigotes of L. amazonensis. The results revealed that compounds (4-6) exhibited higher potency than miltefosine (IC 50 3.11 μM), showing IC 50 values from 0.64 to 2.13 μM. In fact, sesquiterpene lactone 4was 4.8-fold more potent than the reference drug. Besides, the three compounds showed higher selectivity index than miltefosine (Table 3), highlighting compound 6with a SI of 181.81 versus 23.16 for miltefosine. To the best of our knowledge, only one investigation described the leishmanicidal effect of I. viscosa where series of inuloxin derivatives were evaluated against promastigotes of L. donovani [40]. Similar to our finding, compound 4displayed important antiparasitic activity (6.89 μM). Based on literature, there are no investigations that reported the activity of this compound against neither amastigote form nor other Leishmania spp. or T. cruzi. In addition, the characterization of the sesquiterpene lactone 3in I. viscosa is described for the first time and when isolated from other plant species, it was reported as the most active against T. cruzi and L. donovani [41,42]. Furthermore, the results obtained in the current study confirm the noteworthy leishmanicidal and trypanosomal potency of compounds 5and 6commonly found in I. Fig. 6. The effect of inuloxin A (B,G,L), sakuranetin (C,H,M), 8-epi-xanthatin-1β,5β-epoxide (D,I,N), taxifolin (E,J,O) on the mitochondrial potential of Leishmania amazonensis promastigotes compared to the control (A,F,K). JC-1 dye accumulates in the mitochondria of healthy cells as aggregates (red fluorescence) in cells treated with the IC 90 of compounds for 24 h, due to collapse of mitochondrial potential, the JC-1 dye remained in the cytoplasm in its monomeric form, green fluorescence. Images (40X) are representative of the parasites observed in the performed experiments using an EVOS FL Cell Imaging System. The bar graph includes the calculated values of RFU (Fluorescence intensity). Differences between the values were assessed using one-way analysis of variance (ANOVA). Data are presented as means ± SD (N = 3) and letters a-e or A*-E* reflect that means within compounds with different letters are significantly different (p< 0.05). (For interpretation of the references to colour in the Figure, the reader is referred to the web version of this article). Fig. 7. Images (40X) obtained from EVOS FL Cell Imaging System showing the effect of IC 90 of inuloxin A (B,G,L), sakuranetin (C,H,M), 8-epi-xanthatin-1β,5βepoxide (D,I,N), taxifolin (E,J,O) on the mitochondrial potential of Trypanosoma cruzi epimastigotes compared to the control (A,F,K) using JC1 kits after 24 h of incubation. The bar graph includes the calculated values of RFU (Fluorescence intensity). Differences between the values were assessed using one-way analysis of variance (ANOVA). Data are presented as means ± SD (N = 3) and letters a-e or A*-E* reflect that means within compounds with different letters are significantly different (p< 0.05). I. Zeouk, et al. Biomedicine & Pharmacotherapy 130 (2020) 110518 7 Fig. 8. Leishmania donovani (40X) stained with JC-1 kits after 24 h incubation of promastigotes with IC 90 of inuloxin A (B,E,H) and sakuranetin (C,F,I). Images (40X) are representative of the effect on the mitochondrial potential of the parasites and observed using an EVOS FL Cell Imaging System. A control without treatment has been used (A,D,G). The bar graph includes the calculated values of RFU (Fluorescence intensity). Differences between the values were assessed using one-way analysis of variance (ANOVA). Data are presented as means ± SD (N = 3) and letters a-e or A*-E* reflect that means within compounds with different letters are significantly different (p< 0.05). Fig. 9. The effect of 8-epi-xanthatin-1β,5β-epoxide (3), inuloxin A (4), sakuranetin (5) and taxifolin (6) on the ATP production of Leishmania amazonensis (A), Leishmania donovani (B) and Trypanosoma cruzi (C), using CellTiter-Glo Luminescent Cell Viability Assay. Results are representing in percentage relative to the negative control. Cells were treated by the IC 90 concentration for 24 h. Values are given as mean ± SD (N = 3). a-e: means within compounds with different letters are significantly different (p< 0.05). I. Zeouk, et al. Biomedicine & Pharmacotherapy 130 (2020) 110518 8 viscosa [43,44]. The influence of the substitution pattern in the sesquiterpene and flavonoid skeleton on the antikinetoplastid activity was studied, revealing the following trends on the preliminary structure-activity relationship of these natural compounds. In one hand, the influence of the substitution pattern in the sesquiterpene scaffold on the antikinetoplastid activity seems to be linked to the lactone ring (3and 4 versus 1and 2) which is a critical functional group for the activity. Previous data demonstrated the essential function of furanone ring in the biological activities [40,45]. On the other hand, the SAR analysis of the flavonoids showed that the overall oxidation level are important trends of this series, compound 6containing five oxygenated positions has increased potency compared to their parent compounds 5and 8. Moreover, the acetylation and methylation revealed detrimental effect on the activity, when comparing effect of compound 6to that of compound 7. These results highlighted that the best functional group is the hydroxyl, which can act as an H-bond donor suggesting that the hydrophilicity of the molecule contributes to its biological activity. Previous investigations confirmed that the hydrophobic character of molecules decreases the cell permeability of the target reflecting a weak activity [46]. In the same vein, it was suggested that the association of hydroxyl group at C-4′associated to one methoxyl group at C-7 in compound 5is necessary for the antiparasitic potency [47]. The presence of such groups could constitute interesting sites of interaction and/or reaction related to the antiparasitic activity. Furthermore, characteristics such as acetylation were involved in a better transport and distribution in the biological system which enhanced the leishmanicidal effect [48]. 3.2. Programmed cell death pathway Results obtained from the in vitro antiparasitic activities and the selectivity index of compounds over amastigote stage (Tables 2 and 3), were encouraging to analyze their possible mechanism of action. (Table 4). Indeed, cell death of different developmental stages of kinetoplastids such as Trypanosoma and Leishmania genus were coupled to the occurrence of apoptotic events [20]. Among the most relevant apoptotic phenotypes of Leishmania spp. and Trypanosoma spp., there are DNA fragmentation, cell shrinkage, plasma membrane modifications and mitochondrial depolarization [21,49]. Hence, in the present work, different experiments were investigated in order to obtain insights into the apoptotic potential of the four active compounds against L. donovani,L. amazonensis and T. cruzi. We first analyzed the DNA condensation that provides evidence of death through apoptosis. The properties of the performed assay indicate that a normal state of chromatin allows light blue when cells stained with Hoechst 33,342, while the early stained apoptotic cells show bright blue nuclei corresponding to chromatin condensation. While, PI was used to stain the dead cells showing dense bright red nuclei. The staining pattern resulted from this kit leads to distinguish normal, apoptotic and dead parasites. As displayed in Figs. 3,4,5, the treatment of L. amazonensis with compounds 3–6induced a highly noticeable condensation of chromatin, compared with the control (p< 0.05). Moreover, the red fluorescence indicated that PI penetrated the cell Fig. 10. Images (40X) presenting the effects of IC 90 concentration of inuloxin A (B, G), sakuranetin (C, H), 8-epi-xanthatin-1β,5β-epoxide (D, I) and taxifolin (E, J) on the plasma membrane permeability of Leishmania amazonensis promastigotes after 24 h incubation. Cells were labeled with Sytox®Green. Images were obtained using an EVOS FL Cell Imaging System and untreated strains were used as negative control (A, F). The bar graph includes the calculated values of RFU (Fluorescence intensity). Differences between the values were assessed using one-way analysis of variance (ANOVA). Data are presented as means ± SD (N = 3) and letters a-e reflect that means within compounds with different letters are significantly different (p< 0.05). (For interpretation of the references to colour in the Figure, the reader is referred to the web version of this article). I. Zeouk, et al. Biomedicine & Pharmacotherapy 130 (2020) 110518 9