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

Con en s lis s a ailable a ScienceDi ec Biomedicine & Pha maco he apy jou nal homepage: www.else ie .com/loca e/biopha O iginal a icle Sesqui e penoids and fla onoids om Inula iscosa induce p og ammed cell dea h in kine oplas ids Ik ame Zeouk a,b,c, *, Ines Si aoui a,b,d , A ene i López-A encibia a,b,d , Ma ía Reyes-Ba lle a,b,d , Ca los J. Be hencou -Es ella a,b,d , Isabel L. Bazzocchi e , Khadija Bekh i c , Jacob Lo enzo-Mo ales a,b,d , Ignacio A. Jiménez e , José E. Piñe o a,b,d a Ins i u o Uni e si a io de En e medades T opicales y Salud Pública de Cana ias, Uni e sidad de La Laguna, A da. As o ísico Fco. Sánchez, S/N, La Laguna, Tene i e, Islas Cana ias 38203, Spain b Red de In es igación Colabo a i a en En e medades T opicales (RICET) c Depa emen o Biology, Sidi Mohamed Ben Abdellah Uni e si y, Facul y o Sciences and Techniques, Labo a o y o Mic obial Bio echnology and Bioac i e Molecules, PB 2202, Fez, Mo occo d Depa amen o de Obs e icia, Ginecología, Pedia ía, Medicina P e en i a y Salud Pública, Toxicología, Medicina Legal y Fo ense y Pa asi ología, Uni e sidad De La Laguna, La Laguna, Tene i e, Islas Cana ias 38203, Spain e Ins i u o Uni e si a io de Bio-O gánica An onio González, Depa amen o de Química O gánica, Uni e sidad de La Laguna, A enida As o ísico F ancisco Sánchez 2, 38206 La Laguna, Tene i e, Spain ARTICLE INFO Keywo ds: Inula iscosa Leishmanicidal T ypanocidal Sesqui e penoids Fla onoids P og ammed cell dea h ABSTRACT Neglec ed opical diseases such as leishmaniasis and Ame ican ypanosomiasis ep esen an inc easing heal h p oblem. Cu en ea men s a e no sa is ac o y which emains an u gen need o no el, cheap and sa e che- mo he apies. In he cou se o ou ongoing sea ch o new po en ial an i-p o ozoal agen s, his s udy aimed o pe o m a bio-guided ac iona ion o Inula iscosa (As e aceae) using in i o assays agains h ee s ains o Leishmania and T ypanosma genus. Eigh known compounds we e iden ified om he e hanolic ex ac o lea es, sesqui e penoids (3and 4) and fla onoids (5and 6) we e cha ac e ized as he main bioac i e cons i uen s. Sesqui e pene lac ones 3and 4(IC 50 alues be ween 4.99 and 14.26 μM) showed p omising an ipa asi ic ac i i y agains p omas igo es o L. dono ani,L. amazonensis and epimas igo es o T. c uzi. Thei s uc u es we e suc- cess ully cha ac e ized by spec oscopic echniques including 1D and 2D NMR expe imen s. Fu he mo e, he main bioac i e compounds 4,5and 6displayed highe po ency (IC 50 alues be ween 0.64 and 2.13 μM) agains amas igo es o L. amazonensis han mil e osine (IC 50 3.11 μM), and a low oxici y on mac ophages cell line (SI > 45). The analysis o s uc u e-ac i i y ela ionship (SAR) o he an i-p o ozoal ac i i y e ealed ha lac oniza ion o oxida ion enhanced he biological p ofile, sugges ing ha he hyd ophobic moie y was p e- sumably in ol ed in he ac i i y by inc easing he affini y and/o cell memb ane pe meabili y. In o de o ge an insigh in o he mechanism o ac ion o hese compounds, p og ammed cell dea h (PCD) expe imen s we e pe o med, and he ob ained esul s sugges ha he epo ed compounds induced PCD in he ea ed pa asi es. These esul s highligh ha sesqui e penoids and fla onoids om I. iscosa could cons i u e an in e es ing scaffold o he de elopmen o no el an ikine oplas id agen s. 1. In oduc ion Pa asi ic diseases caused by kine oplas ids such as Leishmania and T ypanosoma species, known as neglec ed opical diseases (NTDs), a e esponsible o high mo ali y, disabili y and mo bidi y a es [1]. Ac- co ding o Wo ld Heal h O ganiza ion (WHO), Ame ican ypanoso- miasis, also known as Chagas disease, is a po en ially li e- h ea ening illness caused by he p o ozoan pa asi e T ypanosoma c uzi wi h ap- p oxima ely mo e han 10 000 dea hs pe yea , and mo e han 25 million people a e a a highe isk o acqui ing his disease [2]. Fu - he mo e, leishmaniasis is a complex in ec ious disease wi h a a ied spec um o clinical mani es a ions, which ange om sel -healing cu- aneous ulce a ion o p og essi e and le hal isce al in ec ion [3]. I is p e alen in mo e han 98 endemic coun ies in he wo ld wi h an h ps://doi.o g/10.1016/j.biopha.2020.110518 Recei ed 9 May 2020; Recei ed in e ised o m 24 June 2020; Accep ed 2 July 2020 ⁎ Co esponding au ho a : Ins i u o Uni e si a io de En e medades T opicales y Salud Pública de Cana ias, Uni e sidad de La Laguna, A da. As o ísico Fco. Sánchez, S/N, La Laguna, Tene i e, Islas Cana ias 38203, Spain. E-mail add ess: [email p o ec ed] (I. Zeouk). Biomedicine & Pha maco he apy 130 (2020) 110518 A ailable online 13 July 2020 0753-3322/ © 2020 The Au ho s. Published by Else ie Masson SAS. This is an open access a icle unde he CC BY-NC-ND license (h p://c ea i ecommons.o g/licenses/by-nc-nd/4.0/). T es ima ed annual incidence o almos 0.2 o 0.4 million new cases o isce al mani es a ion and 0.7–1.2 million new cases o cu aneous o m [4]. The eme gence o esis ance, oxici y and high cos o he cu en ea men e eal he u gen need o al e na i e chemo he apeu ic agen s. In his con ex , mo e in e es has been gi en o he de elopmen o new efficien d ugs using science ad ances such as nano echnology [5], chemical syn hesis [6] and na u al p oduc s [7]. Among hese app oaches, plan s p esen a ich sou ce o bioac i e compounds [8]. Fo ins ance, As e aceae amily has mainly p esen ed p omising an i- pa asi ic ac i i ies agains Leishmania and T ypanosoma genus [9]. Be- longing o his amily, Inula iscosa also known as Di ichia iscosa is a medicinal pe ennial he b na i e o he Medi e anean basin and has been widely used in adi ional medicine o ea diffe en diseases [10].P e ious chemical in es iga ions ha e cha ac e ized a ious phy- ochemicals in I. iscosa such as fla onoids, sesqui e penes lac ones, acids and glycolipids wi h a wide ange o he apeu ic indica ions [11–13]. Indeed, fla onoids and sesqui e pene lac ones a e a g oup o na u al p oduc s widely desc ibed as po en an ipa asi ic agen s [14,15]. Recen ly, que ce in ela ed compounds ha e a ge ed he a - ginase in Leishmania amazonensis showing a po en ac i i y [16], and a emisinin which is an illus a i e example o sesqui e pene lac ones, has displayed an ipa asi ic po ency [17] h ough he induc ion o apop o ic-like cell dea h [18,19]. Indeed, cell dea h o diffe en de elopmen al s ages o kine oplas- ids om T ypanosoma and Leishmania genus ha e been coupled o he occu ence o apop o ic ma ke s such as DNA agmen a ion, cell sh inkage, ch oma in condensa ion, loss o mi ochond ial memb ane po en ial ΔΨ and memb ane blebbing [20–22]. Hence, in he p esen wo k we epo a bio-guided ac iona ion o I. iscosa e hanolic ex ac in o de o cha ac e ize leishmanicidal and ypanosomal compounds, o elucida e hei mechanism o ac ion a - ge ing he PCD, and o analyze he s uc u e-ac i i y ela ionship (SAR) in ol ed in hei an i-p o ozoal ac i i y. 2. Ma e ials and me hods 2.1. Gene al p ocedu e, chemicals and eagen s Op ical o a ions we e de e mined on a Pe kin-Elme 241 au oma ic pola ime e . NMR spec a, ROESY (spin lock field 2500 Hz), HSQC and HMBC (op imized o J =7.7 Hz) we e pe o med on a B uke A ance 500 and 600 spec ome e s a 300°K. Silica gel 60 (pa icle size 15–40 and 63–200 μm, Mache ey-Nagel) was used o column ch oma o- g aphy, while silica gel 60 F254 (Mache ey-Nagel) was used o ana- ly ical o p epa a i e TLC and Sephadex LH-20 (Sigma-Ald ich) we e used o he column ch oma og aphic (CC) sepa a ion. Cen i ugal p epa a i e TLC was pe o med using a Ch oma o on (Ha ison Resea ch Inc. model 7924 T) on 4- o 1-mm silica gel 60 PF254 disks wi h flow a e 2−4 mL min −1 . The spo s we e isualized by UV ligh and hea ing silica gel pla es sp ayed wi h H 2 OH 2 SO 4 -AcOH (1:4:20). All he used sol en s we e analy ical g ade om Pan eac. Reagen s, deu - e a ed sol en s we e pu chased om Sigma-Ald ich, benznidazole om Ald ich and mil e osine om Æ e na Zen a is. Fo biological es s, Schneide ’s medium (Sigma-Ald ich), RPMI 1640 and LIT media (Gibco®), alama Blue® eagen (In i ogen, Li e Technologies), EnSpi e®Mul imode Pla e Reade (Pe kin Elme ), and Leika DMIL in- e ed mic oscope (Leika, We zla , Ge many) we e used. 2.2. Plan ma e ial iden ifica ion and ex ac ion In he p esen wo k, he s udied plan s ha e been selec ed om an e hnopha macological s udy unde aken in he cen al no h o Mo occo. The used pa s o he mos ecommended plan s as desc ibed by he balis s ha e been collec ed in he A las Moun ains, Imouzze egion-Mo occo in July 2017, hen iden ified by bo anis s. In o de o ob ain he c ude ex ac s, cleaned used pa s o he diffe en plan s we e shade d ied a oom empe a u e and subse- quen ly milled wi h a simple comme cial elec ic g inde . Powde ed ma e ial was ex ac ed by mace a ion in e hanol (1:10 w/ ) o 6 h a oom empe a u e wi h con inuous s i ing a 500 pm ( e olu ions pe minu e). The esul ing mix u e was fil e ed using Wha man fil e n°1 and he sol en was concen a ed unde acuum in a o a y e apo a o a 45 °C, d ied ex ac s we e s o ed in a e ige a o a 4 °C un il u he use. Amongs he six een es ed plan s, I. iscosa ex ac (5 g, 5%) was he mos ac i e agains he h ee e alua ed pa asi es; he e o e, i was selec ed o u he bioassays. The geog aphical coo dina es o his species a e 33°55′37 N, 5°2′50 W and a ouche specimen (RAB107342) has been deposi ed in he He ba ium o he Scien ific Ins i u e o Raba , Mo occo. 2.3. Bio-guided ac iona ion p ocedu e The p epa ed E OH ex ac s we e assayed o hei an ip o ozoal ac i i y agains p omas igo es o L. amazonensis and L. dono ani and epimas igo es o T. c uzi. A e ha , a bio-guided ac iona ion me ho- dology was used o iden i y an ip o ozoal compounds in he e hanolic ex ac o I. iscosa as he mos ac i e plan . Thus, he ac i e E OH ex ac (5 g) was subjec ed o CC on silica gel elu ed wi h mix u es o hexane-E OAc (100:0 o 0:100, 0.5 L each one) o inc easing pola i y o affo d wel e ac ions ha we e combined in o se en ac ions (F1-F7) based on hei TLC p ofile. The an ip o ozoal ac i i y was ocused on he ac i e ac ions F2 (670.6 mg), F3 (871.5 mg) and F4 (1.7155 g), which we e subjec ed o column ch oma og aphy on sephadex LH-20 column, using a MeOH−CHCl 3 sys em eluen (1:1, 2 L) o affo d be- ween o y and fi y six sub- ac ions which we e combined again based on hei TLC p ofiles (F2/1 o F2/5), (F3/1 o F3/6) and (F4/1 o F4/12), espec i ely. Indeed, he sub- ac ions F3/1 (638.84 mg) and F3/2 (103.56 mg) showed a p omising ac i i y agains he h ee es ed s ains and we e subjec ed o mul iple ch oma og aphic s eps on silica gel, in ol ing medium-p essu e liquid ch oma og aphy, cen i ugal plana ch oma og aphy and p epa a i e TLC using mix u es o hexane- E OAc, hexane-E 2 O, CH 2 Cl 2 -E OAc and CH 2 Cl 2 -ace one as eluen o yield compounds 3(1.78 mg), 4(38.5 mg), 5(3.6 mg) and 7(1.95 mg). Following he same pu ifica ion p ocedu es, ac ion F2 yielded com- pounds 1(3.3 mg) and 3, while ac ion F4 affo ded compounds 2 (8.5 mg), 6(28.5 mg) and 8(5.4 mg). 2.4. Leishmanicidal and ypanocidal assays 2.4.1. Pa asi e s ains The an ipa asi ic ac i i y o c ude ex ac , ac ions and pu e com- pounds o I. iscosa was e alua ed agains he p omas igo e s age o Leishmania dono ani (MHOM/IN/90/GE1F8R), p omas igo e and amas igo e o ms o Leishmania amazonensis (MHOM/BR/77/LTB0016) and epimas igo e o m o T ypanosoma c uzi (Y s ain). 2.4.2. In i o an ileishmanial e alua ion 2.4.2.1. An i-p omas igo es assay. The bioassay was pe o med using he alama Blue®me hod as p e iously desc ibed [23]. P omas igo es o L. dono ani and L. amazonensis we e g own a 26 °C in RPMI 1640 modified medium (Gibco) and supplemen ed wi h 10 % hea - inac i a ed oe al bo ine se um. Cul u es in he loga i hmic phase we e seeded in s e ilized 96-well mic o i e pla es (Co ning™) (10 6 pa asi es/mL) con aining he samples dissol ed in dime hyl sul oxide 1% (DMSO) a he sui able concen a ion o be es ed in se ial dilu ions using Leishmania medium (RPMI 1640) o ge a final olume o 200 μL pe well, hen 20 μL o alama Blue®we e added o he en i e pla e. A e an incuba ion o 72 h, he pla e was checked up isually using an in e ed mic oscope, hen analyzed by an EnSpi e mul imode pla e eade (Pe kinElme , MA, USA) using a es wa eleng h o 570 nm and a e e ence wa eleng h o 630 nm. Leishmanicidal ac i i y was exp essed as IC 50 alues ( he concen a ion o a sample which caused I. Zeouk, e al. Biomedicine & Pha maco he apy 130 (2020) 110518 2 a 50 % educ ion in pa asi e iabili y). Those alues we e calcula ed by linea eg ession analysis wi h 95 % confidence limi s. 2.4.2.2. An i-amas igo es assay. The ac i e pu e compounds om I. iscosa we e es ed agains he in a-mac ophagic s age o L. amazonensis as p e iously desc ibed in li e a u e [24]. In a 96-well fla bo om pla e, he J774A.1 cell line mac ophages we e cul u ed a a densi y o 2 × 10 5 cells/mL in RPMI 1640 medium supplemen ed wi h 10 % hea -inac i a ed oe al bo ine se um and incuba ed o one hou a 37 °C in a 5 % CO 2 a mosphe e in o de o allow almos comple e a achmen o he cells. A e ha , mac ophages we e in ec ed wi h L. amazonensis p omas igo es in he s a iona y phase (7 days old cul u e) wi h a a io o 1:10 (mac ophage/ pa asi e) a a concen a ion o 2×10 6 cells/mL, hen incuba ed a 37 °C in 5% CO 2 o 24 h. Wells we e washed wi h medium o emo e non-phagocy osed p omas igo es. The ea e , he in ec ed mac ophages we e ea ed wi h he pu e compounds (dissol ed in DMSO a he desi ed concen a ion) o 24 h. The medium was emo ed ca e ully o be eplaced by 30 μLo 0.05 % SDS and he pla e was shaken o 30 s. Subsequen ly, 170 μLo Schneide ’s medium we e added o each well o gi e a final olume o 200 μL and 20 μL o alama Blue®we e added o he pla e and incuba ed a 26 °C o 72 h in o de o gi e enough ime o he ans o ma ion om p omas igo es o amas igo es. A e 72 h o incuba ion, pla es we e analyzed using he same p o ocol o p omas igo es es . 2.4.3. In i o e alua ion on T. c uzi epimas igo es The ex ac , ac ions and pu e molecules we e es ed agains he epimas igo es o T. c uzi. B iefly, in 96-well pla es samples dissol ed in DMSO we e se ially dilu ed in 100 μL o LIT medium supplemen ed wi h 10 % hea -inac i a ed e al bo ine se um o ob ain he desi e concen a ions selec ed om he fi s sc eening. In all es s, 1 % DMSO was used o dissol e he highes dose o he compounds wi hou indu- cing any effec s on he pa asi es. A e ha , epimas igo es in loga- i hmic g ow h phase we e coun ed, adjus ed o 5 × 10 5 cells/mL, dis ibu ed in he p e ious 96-well pla e and incuba ed a 27 °C o 72 h. The pla e was obse ed unde an in e ed mic oscope a e 72 h o incuba ion and analyzed s a is ically as desc ibed in he leishmanicidal es . 2.5. Assessmen o cy o oxici y on mac ophages The cy o oxici y assay o he pu e ac i e compounds was pe o med as desc ibed in li e a u e [25]. B iefly, 2 × 10 5 o mac ophages cul u e we e placed in a 96-well pla e o 2 h in a 5 % CO 2 incuba o a 37 °C, hen se ial dilu ions o he 4 ac i e molecules we e p epa ed in deep well, hen ansmi ed o he pla e con aining he cul u e o mac o- phages gi ing a final olume o 100 μL. Finally, he pla e was incuba ed in 5 % CO 2 a mosphe e o 24 h. The pe cen age o cell iabili y was e alua ed using he alama Blue®assay (10 %). Dose esponse cu es we e plo ed and he CC 50 alues we e calcula ed. 2.6. Mechanisms o cell dea h In o de o analyze he p og ammed cell dea h pa hway induced in he s udied pa asi es, a common s ep be ween he ou ki s was pe - o med. B iefly, pa asi es we e ea ed wi h he es ed compounds a hei IC 90 o 24 h. A e ha , cells we e cen i uged (1500 pm o 10 min), washed wice wi h PBS (phospha e buffe ed saline) and in- cuba ed wi h he sui able eagen o each ma ke as desc ibed below. Fo he ou assays, an un ea ed con ol was used. A e incuba ions, an EVOS FL Cell Imaging Sys em AMF4300, Li e Technologies, Mad id, Spain was used o obse e and analyze he ob ained esul s in o de o sco e and o coun cells. 2.6.1. Ch oma in condensa ion de e mina ion To analyze he compac ed s a e o ch oma in in apop o ic cells, a double-s ain apop osis de ec ion ki (Hoechs 33342/PI) (Li e Technologies) was used as ecommended by he manu ac u e . The s ains we e incuba ed wi h Hoechs 33342 a 5 μg/mL and PI a 1 μg/ mL. A e 15 min o incuba ion a 26 °C, samples we e analyzed. 2.6.2. Analysis o mi ochond ial memb ane po en ial The JC-1 Mi ochond ial Memb ane Po en ial Assay Ki (Cayman Chemical) was used o measu e he collapse o an elec ochemical g adien ac oss he mi ochond ial memb ane. T ea ed p omas igo es e- suspended in JC-1 buffe we e incuba ed wi h JC-1 eagen (1:10 / ) and hen incuba ed a 26 °C o 30 min. Analysis o mean g een and ed fluo escence in ensi y and depola iza ion o he mi ochond ial mem- b ane po en ial was analyzed. 2.6.3. De e mina ion o ATP le el ATP le el was measu ed using a Cell Ti e -Glo®Luminescen Cell Viabili y Assay (P omega). The effec o he compounds on he ATP p oduc ion was e alua ed by incuba ing pa asi ic s ains (10 6 cells/mL) wi h he es ed molecules a hei IC 90 o 24 h. 2.6.4. Plasma memb ane pe meabili y To de ec he plasma memb ane pe meabili y o pa asi es, he SYTOX®G een assay was pe o med. The ea ed pa asi es we e in- cuba ed wi h SYTOX®G een a a final concen a ion o 1 μM (Molecula P obes) o 15 min in he da k a oom empe a u e. The inc ease in fluo escence due o binding o he fluo escen ma ke o he pa asi ic DNA was obse ed. 2.6.5. Oxida i e s ess CellRox Deep Red Oxida i e S ess Reagen (The mo Fishe Scien ific) was used o measu e he gene a ion o Reac i e Oxygen Species (ROS) in cells exhibi ing s ong fluo ogenic signal unde oxi- da i e s a e. The assay in ol es he incuba ion o he ea ed pa asi es wi h 5 μM o CellRox Reagen o 30 min a 26 °C, hen he cen- i uga ion o cells o be e-suspended in buffe . H 2 O 2 a 600 μM o 30 min was used as posi i e con ol. The signal o Deep Red is localized in he cy oplasm. Table 1 Leishmanicidal and ypanosomal ac i i y agains p omas igo es o L. amazo- nensis and L. dono ani and epimas igo es o T. c uzi o he ex ac , ac ions and sub- ac ions om lea es o I. iscosa. Samples L. amazonensis IC 50 (μg/mL) L. dono ani IC 50 (μg/mL) T. c uzi IC 50 (μg/mL) C ude ex ac 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 Mil e osine 2.64 ± 0.10 1.35 ± 0.11 – Benznidazole –– 1.81 ± 0.50 IC 50 : Inhibi o y Concen a ion ha inhibi s 50 % o he g ow h o he es ed pa asi e. IC 50 : Means ±S anda d de ia ion. F ac ions and sub- ac ions ha showed IC 50 > 100 μg/mL we e excluded. I. Zeouk, e al. Biomedicine & Pha maco he apy 130 (2020) 110518 3 2.7. S a is ical analysis All assays we e ca ied ou in iplica e. The esul s we e defined as he mean alues o h ee expe imen s. The ob ained inhibi ion cu es was pe o med using he Sigma Plo 12.0 so wa e p og am (Sys a So wa e Inc.). S a is ical analyses we e pe o med using he G aphPad P ism 8.0.2. Fluo escence in ensi y (RFU) was pe o med using ImageJ so wa e p og am as he means alues o h ee epe i ions. Diffe ences be ween he alues we e assessed using a one-way analysis o a iance (ANOVA). Da a a e p esen ed as means ± SD and p< 0.05 was conside ed s a is ically significan . Fig. 1. Flowcha o an ip o ozoal bio-guided ac iona ion o Inula iscosa lea es agains p omas igo es o L. amazonensis and L. dono ani and epimas igo es o T. c uzi (Tc). IC 50 : Inhibi o y concen a ion ha inhibi s 50 % o he g ow h o he es ed pa asi e. IC 50 alues in μg/mL. Fig. 2. Chemical s uc u e o na u al compounds 1-8isola ed om lea es o Inula iscosa. I. Zeouk, e al. Biomedicine & Pha maco he apy 130 (2020) 110518 4 3. Resul s and discussion 3.1. An ikine oplas id ac i i y Resea ch on an ikine oplas id d ugs has been in ensified in ecen yea s exploi ing na u al p oduc s om medicinal plan s [26,27]. The leishmanicidal and ypanosomal ac i i ies o plan ex ac s ha e been a ibu ed o se e al compounds belonging o diffe en chemical g oups such as mono e penes, i e penes, alkaloids, lignans and fla onoids [28–30]. In special, sesqui e penoids and fla onoids isola ed om a ious plan s ha e widely exhibi ed a p ominen leishmanicidal [31] and ypanocidal ac i i y [32]. In he p esen wo k, e hanolic ex ac s o six een plan s used in adi ional medicine o ea men o in ec ious diseases such as skin diso de s, in he cen al no h o Mo occo, we e e alua ed. Roo s o Alkanna in o ia,Be be is hispanica,Ephed a al issima and Rubia inc o ium; ae ial pa s o C a aegus oxyacan ha and U ica dioica; lea es o Ammi majus, Globula ia alypum,Inula iscosa,La andula den a a,Ne ium oleande ,O iganum majo ana, and Rhamnus ala e nus; seeds o E uca sa i a and Junipe us oxyced us and peel o Punica g an- a um. The ex ac s we e fi s ly sc eened a 400, 200, 100; 50 and 25 μg/ mL agains p omas igo e s age o L. dono ani and L. amazonensis, and epimas igo e o m o T. c uzi. The c ude e hanolic ex ac o I. iscosa showed he mos in e es ing ac i i y agains he es ed s ains, wi h IC 50 s be ween 10.87 and 18.10 μg/mL (Table 1) which was a p omising ac o con inue wi h he bioassay-guided ac iona ion in o de o isola e and iden i y he main ac i e molecules in ol ed in he an i- pa asi ic effec s. The e o e, he e hanolic ex ac was submi ed o li- quid ch oma og aphy on silica gel affo ding six ac ions, F1-F6 (Fig. 1). Indeed, samples ha led o a pe cen age o g ow h inhibi ion (%GI) > 50 % we e assayed in diffe en concen a ions o de e mine he hal maximal inhibi o y concen a ion (IC 50 ) and we e classified as highly ac i e (IC 50 <25μg/mL), ac i e (25 < IC 50 <50μg/mL), mode a ely ac i e (50 < IC 50 < 100 μg/mL) and no ac i e when IC 50 > 100 μg/mL. In addi ion, mil e osine and benznidazole we e e alua ed o compa a i e pu poses as e e ence d ugs agains leish- maniasis and Chagas disease espec i ely. Mil e osine showed IC 50 so 2.64 μg/mL and 1.35 μg/mL agains L. amazonensis and L. dono ani, espec i ely, whe eas benznidazole showed an IC 50 o 1.81 μg/mL agains T. c uzi. The IC 50 alues a ied upon he ac ions and he pa asi ic s ains showing diffe en magni udes o he inhibi o y po en ial. The mos ac i e ac ions, F2 (IC 50 alues anging om 8.67 o 35.66 μg/mL), F3 (IC 50 alues anging om 3.80 o 6.61 μg/mL) and F4 (IC 50 alues anging om 3.63 o 27.12 μg/mL), exhibi ed po en ac i i y agains he h ee s udied pa asi es, simila o he e e ence d ugs (Table 1 and Fig. 1), highligh ing hese h ee ac ions as he mos p omising ones. Subsequen ly, hese ac ions we e u he ch oma og aphed on a se- phadex LH-20 column yielding fi e (F2/1-F2/5), six (F3/1-F3/6) and en sub- ac ions (F4/1-F4/10), espec i ely, which we e assayed agains he h ee pa asi es. F om F ac ion 2, F2/4 exhibi ed a po en an ikine oplas id effec on L. amazonensis (IC 50 15.95 μg/mL) and T. c uzi (IC 50 13.74 μg/mL). F om ac ion F3, sub- ac ions F3/1 and F3/2 we e he mos ac i es agains he h ee pa asi es, showing IC 50 s be- ween 6.44 and 12.72 μg/mL o L. amazonensis and L. dono ani, e- spec i ely, and an IC 50 o 9.04 μg/mL on T. c uzi o F3/1.While om ac ion F4, sub- ac ions F4/3, F4/4, F4/5 and F4/6 we e he mos ac i es agains he h ee pa asi es, showing IC 50 s be ween 6.60 and 19.60 μg/mL o L. amazonensis and L. dono ani, espec i ely and IC 50 s be ween 8.59 and 11.18 μg/mL o T. c uzi. The e o e, sub- ac ion F2/ 4 was submi ed o pu ifica ion s eps, affo ding he sesqui e penoids 1 and 3. Addi ionally, sub- ac ion F3/1 showed o be he mos po en one, yielding he compound 3and sub- ac ion F3/2 affo ded Table 2 Leishmanicidal, ypanocidal, cy o oxic ac i i y and selec i i y index agains p omas igo es o Leismania spp., epimas igo es o T. c uzi and mu ine mac ophages o he ac i e sesqui e penoids and fla onoids isola ed om I. iscosa. Compounds L. amazonensis L. dono ani T. c uzi Mu ine mac ophages 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 Mil e osine 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 : Inhibi o y Concen a ion ha inhibi s 50 % o he g ow h o he es ed pa asi e. IC 50 : Means ±S anda d de ia ion. CC 50 : Cy o oxic Concen a ion ha educes 50 % o he mu ine mac ophages’ iabili y. SI: Selec i i y Index (CC 50 /IC 50 ). Compounds ha showed IC 50 > 100 μM we e excluded. Table 3 Leishmanicidal ac i i y and selec i i y index o selec compounds agains amas igo e s age o 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 Mil e osine 3.11 ± 0.29 23.16 IC 50 : Inhibi o y Concen a ion ha inhibi s 50 % o he g ow h o he es ed pa asi e. IC 50 : Means ±S anda d de ia ion. CC 50 : Cy o oxic Concen a ion ha educes 50 % o he mu ine mac ophages’ iabili y. SI a : Selec i i y Index (amas igo es) (CC 50 /IC 50 ). Table 4 Leishmanicidal and ypanocidal ac i i y o he selec ed compounds o s udy o mechanism o ac ion. Compounds IC 90 (μM) L. dono ani L. amazonensis T. c uzi 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 Mil e osine 5.15 ± 0.50 9.63 ± 0.50 – Benznidazole ––25.591 ± 2.728 IC 90 :Effec i e Concen a ion ha inhibi s 90 % o he g ow h o he es ed pa asi e. IC 90 : Means ±S anda d de ia ion. I. Zeouk, e al. Biomedicine & Pha maco he apy 130 (2020) 110518 5 compounds 4,5and 7. Sub- ac ion F4/10 yielded compounds 2,6and 8(Figs. 1,2). Hence, in some cases, he IC 50 was highe han he c ude ex ac which could be explained by a possible syne gism be ween I. iscosa componen s since he complex mix u es o plan componen s may p o ide diffe en ou comes: an addi i e, a syne gis ic o an an- agonis ic effec . The chemical s uc u es o he known isola ed Fig. 3. Leishmania amazonensis p omas igo es incuba ed wi h IC 90 o he ou ac i e compounds o 24 h: Inuloxin A (B,G,L), saku ane in (C,H,M), 8-epi-xan ha in- 1β,5β-epoxide (D,I,N), axi olin (E,J,O). Images (40X) a e ep esen a i e o he cell popula ion obse ed in he pe o med expe imen s using an EVOS FL Cell Imaging Sys em AMF4300, Li e Technologies, USA. Hoechs s ain is diffe en when compa ing nega i e con ol (A,F,K) wi h ea ed cells whe e he nuclei a e b igh blue. Red fluo escence co esponds o he p opidium iodide s ain. Hoechs s ain (F-J), P opidium iodide s ain (K-O). The ba g aph includes he calcula ed alues o RFU (Fluo escence in ensi y). Diffe ences be ween he alues we e assessed using one-way analysis o a iance (ANOVA). Da a a e p esen ed as means ± SD (N = 3) and le e s a-e o A*-E* eflec ha means wi hin compounds wi h diffe en le e s a e significan ly diffe en (p< 0.05). (Fo in e p e a ion o he e e ences o colou in he Figu e, he eade is e e ed o he web e sion o his a icle). Fig. 4. Images (40X) ob ained om an EVOS FL Cell Imaging Sys em showing fluo escence when p omas igo es o Leishmania dono ani incuba ed wi h IC 90 o he ou es ed compounds o 24 h: Inuloxin A (B,G,L), saku ane in (C,H,M), 8-epi-xan ha in-1β,5β-epoxide (D,I,N), axi olin (E,J,O) and s ained wi h Hoechs - p opidium iodide. Hoechs co esponds o he ch oma in condensa ion (blue) in ea ed cells. Red fluo escence co esponds o he p opidium iodide s ain. A con ol wi hou ea men has been used (A,F,K). The ba g aph includes he calcula ed alues o RFU (Fluo escence in ensi y). Diffe ences be ween he alues we e assessed using one-way analysis o a iance (ANOVA). Da a a e p esen ed as means ± SD (N = 3) and le e s a-e o A*-E* eflec ha means wi hin compounds wi h diffe en le e s a e significan ly diffe en (p< 0.05). (Fo in e p e a ion o he e e ences o colou in he Figu e, he eade is e e ed o he web e sion o his a icle). Fig. 5. Images (40X) ob ained om EVOS FL Cell Imaging Sys em showing he effec o Inuloxin A (B,G,L), saku ane in (C,H,M), 8-epi-xan ha in-1β,5β-epoxide (D, I,N), axi olin (E,J,O) a he IC 90 agains epimas igo es o T ypanosoma c uzi a e 24 h o incuba ion in compa ison o he con ol (A,F,K), hen using Hoechs - p opidium iodide double s ains. Hoechs co esponds o he ch oma in condensa ion (blue) in ea ed cells. Red fluo escence co esponds o he p opidium iodide s ain. The ba g aph includes he calcula ed alues o RFU (Fluo escence in ensi y). Diffe ences be ween he alues we e assessed using one-way analysis o a iance (ANOVA). Da a a e p esen ed as means ± SD (N = 3) and le e s a-e o A*-E* eflec ha means wi hin compounds wi h diffe en le e s a e significan ly diffe en (p< 0.05). (Fo in e p e a ion o he e e ences o colou in he Figu e, he eade is e e ed o he web e sion o his a icle). I. Zeouk, e al. Biomedicine & Pha maco he apy 130 (2020) 110518 6 compounds (1-8) we e iden ified as isocos ic acid (1)[33], ilicic acid (2)[34], 8-epi-xan ha in-1β,5β-epoxide (3)[35], inuloxin A (4)[36], saku ane in (5)[37], axi olin (6)[38], 3-O-ace yl-7-O-me hyla - omadend in (7)[38], and que ce in (8)[39] using spec ome ic and spec oscopic da a, including 1D and 2D NMR expe imen s, in addi ion o compa ison wi h da a epo ed in he li e a u e. Compounds 1-8 we e e alua ed agains he h ee pa asi es. In addi ion, he cy o oxici y on mu ine mac ophages was also assessed. Mil e osine showed a CC 50 o 72.18 μM, whe eas benznidazole showed a CC 50 o 399.91 μM (Table 2). The esul s, o e he p omas igo e s age o Leishmania, showed ha compounds 3and 4displayed po en ac i i y in μM ange and we e sligh ly less po en han he e e ence d ug. Mo eo e , com- pounds 3and 4showed simila po ency o benznidazole agains epi- mas igo es o T. c uzi. Rega ding he selec i i y index (SI) owa ds mac ophages (Table 2), he compounds 3and 4showed a mode a ed cy o oxic p ofile on he h ee pa asi es (SI alues anging om 1.66 o 3.67). Based on he in i o esul s on p omas igo e o ms, compounds 3-6we e selec ed o be e alua ed on in acellula amas igo es o L. amazonensis. The esul s e ealed ha compounds (4-6) exhibi ed highe po ency han mil e osine (IC 50 3.11 μM), showing IC 50 alues om 0.64 o 2.13 μM. In ac , sesqui e pene lac one 4was 4.8- old mo e po en han he e e ence d ug. Besides, he h ee compounds showed highe selec i i y index han mil e osine (Table 3), highligh ing com- pound 6wi h a SI o 181.81 e sus 23.16 o mil e osine. To he bes o ou knowledge, only one in es iga ion desc ibed he leishmanicidal effec o I. iscosa whe e se ies o inuloxin de i a i es we e e alua ed agains p omas igo es o L. dono ani [40]. Simila o ou finding, compound 4displayed impo an an ipa asi ic ac i i y (6.89 μM). Based on li e a u e, he e a e no in es iga ions ha epo ed he ac i i y o his compound agains nei he amas igo e o m no o he Leishmania spp. o T. c uzi. In addi ion, he cha ac e iza ion o he sesqui e pene lac one 3in I. iscosa is desc ibed o he fi s ime and when isola ed om o he plan species, i was epo ed as he mos ac i e agains T. c uzi and L. dono ani [41,42]. Fu he mo e, he esul s ob ained in he cu en s udy confi m he no ewo hy leishmanicidal and ypanosomal po ency o compounds 5and 6commonly ound in I. Fig. 6. The effec o inuloxin A (B,G,L), saku ane in (C,H,M), 8-epi-xan ha in-1β,5β-epoxide (D,I,N), axi olin (E,J,O) on he mi ochond ial po en ial o Leishmania amazonensis p omas igo es compa ed o he con ol (A,F,K). JC-1 dye accumula es in he mi ochond ia o heal hy cells as agg ega es ( ed fluo escence) in cells ea ed wi h he IC 90 o compounds o 24 h, due o collapse o mi ochond ial po en ial, he JC-1 dye emained in he cy oplasm in i s monome ic o m, g een fluo escence. Images (40X) a e ep esen a i e o he pa asi es obse ed in he pe o med expe imen s using an EVOS FL Cell Imaging Sys em. The ba g aph includes he calcula ed alues o RFU (Fluo escence in ensi y). Diffe ences be ween he alues we e assessed using one-way analysis o a iance (ANOVA). Da a a e p esen ed as means ± SD (N = 3) and le e s a-e o A*-E* eflec ha means wi hin compounds wi h diffe en le e s a e significan ly diffe en (p< 0.05). (Fo in e p e a ion o he e e ences o colou in he Figu e, he eade is e e ed o he web e sion o his a icle). Fig. 7. Images (40X) ob ained om EVOS FL Cell Imaging Sys em showing he effec o IC 90 o inuloxin A (B,G,L), saku ane in (C,H,M), 8-epi-xan ha in-1β,5β- epoxide (D,I,N), axi olin (E,J,O) on he mi ochond ial po en ial o T ypanosoma c uzi epimas igo es compa ed o he con ol (A,F,K) using JC1 ki s a e 24 h o incuba ion. The ba g aph includes he calcula ed alues o RFU (Fluo escence in ensi y). Diffe ences be ween he alues we e assessed using one-way analysis o a iance (ANOVA). Da a a e p esen ed as means ± SD (N = 3) and le e s a-e o A*-E* eflec ha means wi hin compounds wi h diffe en le e s a e significan ly diffe en (p< 0.05). I. Zeouk, e al. Biomedicine & Pha maco he apy 130 (2020) 110518 7 Fig. 8. Leishmania dono ani (40X) s ained wi h JC-1 ki s a e 24 h incuba ion o p omas igo es wi h IC 90 o inuloxin A (B,E,H) and saku ane in (C,F,I). Images (40X) a e ep esen a i e o he effec on he mi ochond ial po en ial o he pa asi es and obse ed using an EVOS FL Cell Imaging Sys em. A con ol wi hou ea men has been used (A,D,G). The ba g aph includes he calcula ed alues o RFU (Fluo escence in ensi y). Diffe ences be ween he alues we e assessed using one-way analysis o a iance (ANOVA). Da a a e p esen ed as means ± SD (N = 3) and le e s a-e o A*-E* eflec ha means wi hin compounds wi h diffe en le e s a e significan ly diffe en (p< 0.05). Fig. 9. The effec o 8-epi-xan ha in-1β,5β-epoxide (3), inuloxin A (4), saku ane in (5) and axi olin (6) on he ATP p oduc ion o Leishmania amazonensis (A), Leishmania dono ani (B) and T ypanosoma c uzi (C), using CellTi e -Glo Luminescen Cell Viabili y Assay. Resul s a e ep esen ing in pe cen age ela i e o he nega i e con ol. Cells we e ea ed by he IC 90 concen a ion o 24 h. Values a e gi en as mean ± SD (N = 3). a-e: means wi hin compounds wi h diffe en le e s a e significan ly diffe en (p< 0.05). I. Zeouk, e al. Biomedicine & Pha maco he apy 130 (2020) 110518 8 iscosa [43,44]. The influence o he subs i u ion pa e n in he sesqui e pene and fla onoid skele on on he an ikine oplas id ac i i y was s udied, e- ealing he ollowing ends on he p elimina y s uc u e-ac i i y e- la ionship o hese na u al compounds. In one hand, he influence o he subs i u ion pa e n in he sesqui e pene scaffold on he an i- kine oplas id ac i i y seems o be linked o he lac one ing (3and 4 e sus 1and 2) which is a c i ical unc ional g oup o he ac i i y. P e ious da a demons a ed he essen ial unc ion o u anone ing in he biological ac i i ies [40,45]. On he o he hand, he SAR analysis o he fla onoids showed ha he o e all oxida ion le el a e impo an ends o his se ies, compound 6con aining fi e oxygena ed posi ions has inc eased po ency compa ed o hei pa en compounds 5and 8. Mo eo e , he ace yla ion and me hyla ion e ealed de imen al effec on he ac i i y, when compa ing effec o compound 6 o ha o com- pound 7. These esul s highligh ed ha he bes unc ional g oup is he hyd oxyl, which can ac as an H-bond dono sugges ing ha he hy- d ophilici y o he molecule con ibu es o i s biological ac i i y. P e- ious in es iga ions confi med ha he hyd ophobic cha ac e o mo- lecules dec eases he cell pe meabili y o he a ge eflec ing a weak ac i i y [46]. In he same ein, i was sugges ed ha he associa ion o hyd oxyl g oup a C-4′associa ed o one me hoxyl g oup a C-7 in compound 5is necessa y o he an ipa asi ic po ency [47]. The p e- sence o such g oups could cons i u e in e es ing si es o in e ac ion and/o eac ion ela ed o he an ipa asi ic ac i i y. Fu he mo e, cha ac e is ics such as ace yla ion we e in ol ed in a be e anspo and dis ibu ion in he biological sys em which enhanced he leishmanicidal effec [48]. 3.2. P og ammed cell dea h pa hway Resul s ob ained om he in i o an ipa asi ic ac i i ies and he selec i i y index o compounds o e amas igo e s age (Tables 2 and 3), we e encou aging o analyze hei possible mechanism o ac ion. (Table 4). Indeed, cell dea h o diffe en de elopmen al s ages o ki- ne oplas ids such as T ypanosoma and Leishmania genus we e coupled o he occu ence o apop o ic e en s [20]. Among he mos ele an apop o ic pheno ypes o Leishmania spp. and T ypanosoma spp., he e a e DNA agmen a ion, cell sh inkage, plasma memb ane modifica- ions and mi ochond ial depola iza ion [21,49]. Hence, in he p esen wo k, diffe en expe imen s we e in es iga ed in o de o ob ain in- sigh s in o he apop o ic po en ial o he ou ac i e compounds agains L. dono ani,L. amazonensis and T. c uzi. We fi s analyzed he DNA condensa ion ha p o ides e idence o dea h h ough apop osis. The p ope ies o he pe o med assay indica e ha a no mal s a e o ch oma in allows ligh blue when cells s ained wi h Hoechs 33,342, while he ea ly s ained apop o ic cells show b igh blue nuclei co esponding o ch oma in condensa ion. While, PI was used o s ain he dead cells showing dense b igh ed nuclei. The s aining pa e n esul ed om his ki leads o dis inguish no mal, apop o ic and dead pa asi es. As displayed in Figs. 3,4,5, he ea - men o L. amazonensis wi h compounds 3–6induced a highly no ice- able condensa ion o ch oma in, compa ed wi h he con ol (p< 0.05). Mo eo e , he ed fluo escence indica ed ha PI pene a ed he cell Fig. 10. Images (40X) p esen ing he effec s o IC 90 concen a ion o inuloxin A (B, G), saku ane in (C, H), 8-epi-xan ha in-1β,5β-epoxide (D, I) and axi olin (E, J) on he plasma memb ane pe meabili y o Leishmania amazonensis p omas igo es a e 24 h incuba ion. Cells we e labeled wi h Sy ox®G een. Images we e ob ained using an EVOS FL Cell Imaging Sys em and un ea ed s ains we e used as nega i e con ol (A, F). The ba g aph includes he calcula ed alues o RFU (Fluo escence in ensi y). Diffe ences be ween he alues we e assessed using one-way analysis o a iance (ANOVA). Da a a e p esen ed as means ± SD (N = 3) and le e s a-e eflec ha means wi hin compounds wi h diffe en le e s a e significan ly diffe en (p< 0.05). (Fo in e p e a ion o he e e ences o colou in he Figu e, he eade is e e ed o he web e sion o his a icle). I. Zeouk, e al. Biomedicine & Pha maco he apy 130 (2020) 110518 9