OPEN ACCESS Resea ch Jou nal o Medicinal Plan s
ISSN 1819-3455
DOI: 10.3923/ jmp.2018.57.64
Resea ch A icle
An iplasmodial and Cy o oxic Ac i i y o Pipe Piedecues anum
T el. and Yunck
1,2Ana Ma ía Mesa Vanegas, 1Jhon F edy To o Suaza, 1Ana Ma ía Vásquez Ca dona, 1Sil ia Blai T ujillo,
2Ca los Peláez Ja amillo, 3,4San iago Díaz Ol a, 3Césa Augus o Pachón and 3Miguel Ca da
1Mala ia Resea ch G oup, Uni e si y Resea ch Headqua e s (SIU), Ca e a 62 52-59, Towe 1, Lab. 610 SIU, Facul y o Medicine,
Uni e si y o An ioquia, A.A 1226, Medellín, Colombia
2In e disciplina y G oup o Molecula S udies GIEM, Facul y o Exac and Na u al Sciences, Uni e si y o An ioquia, A.A 1226,
Medellín, Colombia
3Depa men o Ino ganic and O ganic Chemis y, Uni e si a Jaume I, 12071, Cas ellón, Spain
4Depa men o Educa ion, Uni e si a Jaume I, 12071, Cas ellón, Spain
Abs ac
Backg oundandObjec i e:Plasmodium esis ance oan imala iald ugshasexpandedandin ensi ied,makingnewande ec i e
an imala iald ugsu gen ly.Theobjec i eo  hiswo kwas he
in i o
e alua iono an iplasmodialac i i yo ex ac so di e en pola i y
andcompoundso  hespecies
P.piedecues anum
.Ma e ialsandMe hods:Theplan ma e ialswe eob ained h oughsuccessi e
ex ac ionsusingsol en so di e en pola i ysuchashexane(H),dichlo ome hane(D),e hylace a e(A)andme hanol(M)and
sepa a ions echniques o  ac iona ionandisola iono compounds.Thean iplasmodialac i i ieso  heex ac sandcompoundswe e
e alua edbySYBRG eenI®me hodande alua ed hecy o oxici yin hecelllinesU-937,HUVECby heMTTme hod.Resul s:The
an iplasmodialandcy o oxicac i i yo  heex ac so dichlo ome hane(PPD)ande hylace a e(PPAE)wi han iplasmodialac i i y
o IC50=17.93µgmLG1;IS=2.093andIC50=19.5µgmLG1;IS=0.791, espec i elya e epo ed o  he i s  ime.Inaddi ion, om
P.piedecues anum
specieswe eisola ionandcha ac e iza ion i eme aboli es5,8-Hyd oxy-7-me hoxy la one(1),6,7-dime hoxy-5,8-
dihyd oxy la one(2),6,7-dime hoxy-5-hyd oxy la one(moslo la one)(3),5,6-dihyd oxy-7-me hoxy la one(negle ein)(4),5-hyd oxy-7-
me hoxy la one(5)andab omina edde i a i e om(5)named6,8b omo-5-hyd oxy-7-me hoxy la one(7).Compound(1)p esen ed
p omisingan iplasmodialac i i ywi hanIC50=7.325µgmLG1(25.69µM);ISHUVEC=13.65.Conclusion:Chemicalanalysiso ex ac sand
compounds om
P.piedecues anum
spices

willplayacen al olein hede elopmen andmode niza iono anan imala ialhe bal
adi ionalinColombia.
Keywo ds: 
Pipe pidecues anum
,an iplasmodialac i i y,cy o oxicac i i y,
Plasmodium alcipa um
Ci a ion: AnaMa íaMesaVanegas,JhonF edyTo oSuaza,AnaMa íaVásquezCa dona,Sil iaBlai T ujillo,Ca losPeláezJa amill,San iagoDíazOl a,Césa
Augus oPachón,MiguelCa da,2018.An iplasmodialandcy o oxicac i i yo pipe piedecues anumT el.andYunck.Res.J.Med.Plan s,12:57-64.
Co espondingAu ho :AnaMa íaMesaVanegas,Uni e si yo An ioquia,Facul yo Exac andNa u alSciences,Ins i u eo Biology,Uni e si yo An ioquia,
A.A1226,Medellín,ColombiaTel:+573017947846
Copy igh : ©2018AnaMa íaMesaVanegas
e al
.Thisisanopenaccessa icledis ibu edunde  he e mso  hec ea i ecommonsa ibu ionLicense,
whichpe mi sun es ic eduse,dis ibu ionand ep oduc ioninanymedium,p o ided heo iginalau ho andsou cea ec edi ed.
Compe ingIn e es : Theau ho sha edecla ed ha nocompe ingin e es exis s.
Da aA ailabili y: All ele an da aa ewi hin hepape andi ssuppo ingin o ma ion iles.
Res.J.Med.Plan s,12(2):57-64,2018
INTRODUCTION
Mala ia is a disease caused by p o ozoan pa asi es
belonging o he amilyPlasmodiidae,genus Plasmodium,
whicha e ansmi edby emalemosqui oeso  hegenus
Anopheles1.Cu en ly,mala iaisap essingheal hp oblemin
many pa s o  he wo ld, pa icula ly in A ica and La in
Ame ica, whicha e he egionswi h hehighes mo ali y
a es.Recen da aindica e ha mala iaisp esen in
97coun iesandanes ima ed3.2billionpeoplea ea  isko
con ac ing hedisease2.In heyea 2015,214millioncaseso
mala iawe e epo edand438.000peoplediedmos o  hem
child enunde 5yea so age.Mos o  hesecasesoccu ed
inA ica3.A p esen , hesi ua ionisbecominge enmo e
complica ed by he sp ead o  d ug esis an  pa asi es,
especially in a eas whe e
Plasmodium alcipa um
and
Plasmodium i ax
a eendemicando highe p e alence.
Few al e na i e d ugs a e unde  de elopmen  and u gen
measu esa e equi ed oiden i ynewclasseso an imala ial
agen s, many o  which ha e hei  o igins om na u al
p oduc s4.
Pipe , henominalgenuso  he amilyPipe aceae,isone
o  hemos di e segene ao basalangiospe ms.I iscu en ly
conside ed o ha e abou  1500 species and i s g ea es
di e si y is ound in he humid o es s o  opical egions
a ound he wo ld5. Ecologically, hey a e impo an  and
dominan componen sin hehumid o es s,especiallyin he
neo opicsand heycons i u eanimpo an pa o  hedie o
some amilies o  Ame ican ba s, insec s and bi ds6-8. Few
specieso Pipe a eeconomically impo an ,among hem
Pipe nig um
 omwhich hepeppe isob ained,condimen
popula lyusedallo e  hewo ld.Fo  hePipe genus,awide
angeo  adi ional oodshasbeen epo edin he ea men
o  a iousdiseasessuchasmala ia,anemia,chole a,diabe es,
as hma,b onchi is,pneumonia,in luenza, heuma ismand
 a h i is. In his s udy, plan s we e used as condimen s,
aph odisiacs,s imulan sandhallucinogens9-11.Chemically,in
Pipe aceae,lignansandneolignansha ebeen ound, annins,
saponins, phenolic compounds, e penes, la onoids and
alkaloids among o he s12. Many o  hese compounds,
especiallyalkaloids, e penesandlignans,a e esponsible
o  hean iplasmodialac i i yo manyspecies epo edin
o he  pa s o  he wo ld13,14. A la ge a ie y o  Pipe aceae
speciesa eusedby adi ionalmedicine o ea mala ia,some
o whichha ebeen hesubjec o an iplasmodialac i i ybo h
in i o
and
in i o
15.Rega ding hean iplasmodialpo en ial
o ex ac sandcomponen so 
P.piedecues anum
T el.and
Yunck., he ea eno  epo sin heli e a u eand he eisa
single epo in es iga ing hean ioxidan ac i i yo  hese
ex ac s16.
Thus, hep esen s udywasaimed oob ainex ac so
di e en pola i yo  hespecies
P.piedecues anum
T el.and
Yunck,isola edandcha ac e ized hei majo componen s.
E alua e an iplasmodial ac i i y
in  i o
  on con inuous
cul u eso 
P. alcipa um
chlo oquine-sensi i es ainNF-54
and e alua ed he cy o oxici y ac i i y o  heex ac s and
compoundsin hecelllinesU-937andHUVEC.
MATERIALSANDMETHODS
Allexpe imen swe e ealizedinLabo a o yo Mala ia
Resea ch G oup. Uni e si y Resea ch Headqua e s (SIU),
Uni e si yo An ioquia,Medellin,ColombiaandDepa men
o  Ino ganic and O ganic Chemis y, Uni e si a  Jaume I,
Cas ellón,Spain.
Chemical and sol en s: hexane, dichlo ome hane, e hyl
ace a e,me hanol,chlo o o m,e hanol,dime hylsulphoxide
(DMSO),Silica-gel60,sul u icacid,N,N-Dime hyl o mamide
(DMF),N-B omosuccinimide(NBS),RPMI-1640,HEPES,
SYBR G een I®, 3- (4,5-dime hyl hiazol-2-yl) -2,5-diphenyl
e azolium b omide (MTT)and s anda d chlo oquine we e
used.Analy icalg ade eagen s/chemicalswe ealsousedin
hisexpe imen .
Collec ionandiden i ica iono plan ma e ial:Th ee ypes
o sampleswe ecollec ed om heplan ma e ial:Asample
o he ba iumspecimen,samplesascon olso specimens
collec ed and samples o  lea es and s ems o ob ain he
ex ac s. The specimen o  he ba ium was p ocessed,
deposi edand axonomicallycha ac e izedin hehe ba ium
o  heUni e si yo An ioquia(HUA)andde e minedas
Pipe piedecues anum
T el.andYunck.(Vouche  .191.1950)
collec edinPiedecues a,San ande -Colombia.
P epa a iono ex ac sandisola iono compounds:
Pipe piedecues anum
(PP)plan ma e ialwassubjec ed o
adesicca ionp ocessa  oom empe a u ewi hae a ionand
wi hou exposu e osunligh  o 10days.Abou 2.36go
mix u eo lea esands emso  heg ound ege ablema e ial
wasini ially aken oape cola ionp ocessun ilexhaus ion
(5days/3 imes)usinge hanol(E) ha was hen il e edand
concen a edona o a ye apo a o .On heo he hand,
0.36kgo g oundma e ialwassubjec ed oex ac ionwi h
sol en so upwa dpola i y(500mL)byape cola ionp ocess
oexhaus ionwi heacho  he ollowingsol en s:hexane(H),
dichlo ome hane(D),e hylace a e(A)andme hanol(M).A e
3days, heex ac wasconcen a edunde  educedp essu e
ina o a ye apo a o .The i eex ac so  heplan we e
58
Res.J.Med.Plan s,12(2):57-64,2018
codedwi h heini ialso  hespeciesnameand he ypeo
ex ac andacco ding o hepola i yo  hesol en ,s a ing
wi h he pe oleum e he  (H) (PPH), dichlo ome hane (D)
ex ac (PPD),e hylace a e(EA)(PPAE),me hanol(M)(PPM)
and e hanolic ex ac  encoded as (PPE). All ex ac s we e
moni o edby hinlaye ch oma og aphy(CCD)suppo ed
wi hMe ck®Silica-gel60GF254usingdi e en elu ionsys ems.
Theex ac ionpe cen ageso  heex ac swe ecalcula ed
acco ding oEq.1:
(1)
Ex ac s weigh
deEx ac ion (%) 100
Weigh o plan ma e ial

Theex ac sconside edasac i ewe ep ocessed
by ac iona ionandisola iono  hemajo subs ances.
Theex ac o dichlo ome hane(PPD)o  hespecieso
P.piedecues anum
T el.andYunck.(4.89g)wasac i eand
was ac iona edbycolumnch oma og aphyusingaseluen
g adien so pe oleume he :e hylace a e,E OAcandMeOH.
Thi y ac ionswe eob ained omwhich ac ion10was
aken and column ch oma og aphy was pe o med using
pe oleume he ,pe oleume he :DCM(1:1),DCM,g adien s
o  DCM: E OAc and inally MeOH as eluen . Twen y h ee
ac ionswe eob ainedwhichwe epooledbe ween1-7and
p epa a i epla ech oma og aphywaspe o medusingDCM
as heeluen and20mgo  hecompounddesigna edas(4)
we e pu i ied. On he o he  hand, o sec ion 25 o  he
dichlo ome hane ex ac  column, p epa a i e laye
ch oma og aphywaspe o medand wocompoundswe e
isola ed,oneo whichwasayellowamo phoussolidnamed
(2)(31.8mg)and heo he ac ys allineo angesolid e e ed
oas(1)(183.9mg).The emaining ac ionso  hepe oleum
e he ex ac anddichlo ome hanewe ecombined ope o m
column ch oma og aphy again using pe oleum e he
g adien s: DCM, DCM: E OAc, E OAc: Me hanol (MeOH)
g adien s and inally MeOH and ob ained 14 ac ions,
ac ionso 6-10we e akenandcolumnch oma og aphy
usingeluen pe oleume he ,pe oleume he :DCM(1:1),
DCM and DCM: MeOH g adien s as eluen s, whe eby 30
ac ions, ac ions1-9and15-17we e aken o p epa a i e
pla ech oma og aphyelu ingwi hpe oleume he :
DCM25:3andDCM oisola e39mgo  hecompound(4),
59mgo compound(3)and58mgo compound
(5). Halogena ed a oms we e in oduced in o he isola ed
compoundsby hep oduc iono b omina edde i a i es.Only
asuccess ul eac ionwaspe o med omcompound(5).
The expe imen al p ocedu e is b ie ly desc ibed below: a
solu iono (5)(20mg,0.71mmol)wasdissol edinN,
N-Dime hyl o mamide(DMF)andN-B omosuccinimideNBS
(0.71mmol)wasadded.The eac ionmix u ewasle a 0EC
o 2hunde ni ogen.Subsequen ly, he empe a u ewas
g adually aised o 80EC o  24 h. The c ude mix u e was
dilu ed in dichlo ome hane (10 mL) and washed wi h
sa u a ed aqueous ammonium chlo ide (3×10 mL). The
o ganic laye  was d ied (Mg2SO4) and concen a ed unde
educed p essu e o gi e compound (6). All ex ac s and
compounds we e s o ed a  oom empe a u e o  he
biologicalassays.
S uc u al cha ac e iza ion: IR spec a we e ob ained by
using KB  pelle s on a Jasco FT/IR-6200 spec ome e ,
spanning he egion 4000-600 cmG1. Mass spec a we e
measu ed on a Q-TOF mass spec ome e  (Wa e s,
Manches e ,UK)wi helec osp ay- ypecombinedioniza ion
sou ceandZ-sp aydesignAPCI; hecapilla y ol ageo 3.5KV
wasusedin heposi i edi ec ionand hecone ol agewas
se  a  20V. NMR spec a we e eco ded on Va ian Uni y
spec ome e s o  300 and 500 (app oxima e ope a ing
equencies,300and500MHz o 1H,125and75MHz o 13C).
The na u e o  he ca bon signals (C, CH, CH2, CH3) was
de e mined using he APT o  DEPT echniques. Signal
assignmen s we e pe o med using wo-dimensional
he e onuclea co ela ions(COSYandHMQC/HMBC).Unless
o he wise indica ed, he spec a we e measu ed in CDCl3
solu ion.Chemicalshi s(δ)a e epo edinppmusing he
esidualsol en signals(δ7.27ppm o 1Hand77.0ppm o
13C)as e e ence.As a as he e e encingo  hespec awhen
i comes omul iple s, he ange heyoccupyisincluded.
Compound (1): 5,8-Hyd oxy-7-me hoxy la one, yellow
amo phoussolid.C16H12O5.TOFMSES+[M+H]:285.0760.
1H-NMR-300MHz δ(chlo o o m-d1)(ppm):12.50(1H,s,
C5-OH),7.89(2H,dd,J=2.2,8.0Hz,A -H),7.56-7.52(3H,m,
A -H),6.69(1H,s,C6-H),6.63(1H,s,C3-H),4.018(3H,s,CH3O).
13C-NMR-75 MHz δ (chlo o o m-d1) (ppm): 183.06, 165.59,
162.15,153.33,146.04,132.19,131.88,130.048,129.49
(CH×2),126.67(CH×2),105.91,105.85,90.95,56.8817.
Compound(2):6,7-dime hoxy-5,8-dihyd oxy la one,yellow
amo phoussolid.C17H14O5.TOFMSES+[M+OH]:315.0869.
1H-NMR-300MHz δ(chlo o o m-d1)(ppm):12.63(1H,s,
C5-OH),8,20(2H,dd,J=2.2,8.0Hz,A -H),7.53-7.51(3H,m,
A -H),6.82(1H,bs,C8-OH),6.56(1H,s,C3-H),3.98(3H,s,CH3O),
3.94(3H,s,CH3O).13C-NMR-75MHzδ(chlo o o m-d1)(ppm):
197.36,174.85,159.77,152.88,151.92,136.96,132.52,130.72,
129.04 (CH×2), 128.03 (CH×2), 96.95, 91.36, 86.65, 61.37,
56.8017.
59
Res.J.Med.Plan s,12(2):57-64,2018
Compound (3): 6,7-dime hoxy-5-hyd oxy la one
(moslo la one),yellowamo phoussolid.C17H14O5.TOFMSES+
[M+H]:299.0919.1H-NMR-500MHzδ(chlo o o m-d1)(ppm):
12.68(1H,s,C5-OH),7.88(2H,dd,J=2.2,8.0Hz,A -H),
7.54-7.52(3H,m,A -H),6.66(1H,s,C3-H),6.56(1H,s,C8-H),
3.97 (3H, s, CH3O), 3.93 (3H, s, CH3O). 13C-NMR-125 MHz δ
(chlo o o m-d1)(ppm):182.66,163.90,158.87,153.27,152.99,
132.66,131.80,131.25,129.05(CH×2),126.20(CH×2),106.24,
105.56,90.62,60.80,56.2917.
Compound(4):5,6-dihyd oxy-7-me hoxy la one(negle ein),
yellow amo phous solid. C16H12O5. TOF MS ES+ [M+H]:
285.0767.1H-NMR-300MHzδ(chlo o o m-d1)(ppm):11.72
(1H,bs,C5-OH),8.20(2H,dd,J=2.2,8.0Hz,A -H),7.53-7.48
(3H,m,A -H),6.51(1H,s,C3-H),6.39(1H,s,C8-H),3.90(3H,s,
CH3O). 13C-NMR-75 MHz δ (chlo o o m-d1) (ppm): 194.62,
166.54,166.0,161.55,145.36,131.97,130.314,129.49,129.46
(CH×2),129.08(CH×2),114.04,104.95,94.04,57.6017.
Compound (5): 5-hyd oxy-7-me hoxy la one, yellow
amo phoussolid.C17H14O5.TOFMSES+[M+H]:269.0810.
1H-NMR-500MHz δ(chlo o o m-d1)(ppm):12.72(1H,s,
C5-OH),7.88(2H,dd,J=2.2,8.0Hz,A -H),7.53-7.52(3H,m,
A -H),6.67(1H,s,C3-H),6.50(1H,s,C6-H),6.37(1H,s,C8-H),3.88
(3H, s, CH3O). 13C-NMR-125 MHz δ (chlo o o m-d1) (ppm):
182.46,165.59,162.15,157.76,131.80,131.28(CH×2),129.05
(CH×2),126.64,105.83,105.67,98.17,92.65,55.7817.
Compound (6,7): 6,8 b omo-5-hyd oxy-7-me hoxy la one,
c ys alyellowcolo .C16H11O4B 2.TOFMSES+[M+H]:426.9007.
1H-NMR-500MHz δ(chlo o o m-d1)(ppm):12.72(1H,s,
C5-OH),7.89(2H,dd,J=2.2,8.0Hz,A -H),7.53-7.52(3H,m,
A -H),6.67(1H,s,C3-H),3.88(3H,s,CH3O)17.
Biologicalac i i y es
In i o
de e mina iono an iplasmodialac i i y:
In i o
an iplasmodialac i i yassayswe epe o medon hesensi i e
(NF-54)chlo oquines ain.
P. alcipa um
s ains(NF-54)we e
cul u edandmain ainedacco ding o heme hodo
T age andJensen18,usingasuspensiono 5%human
A+e y h ocy esinRPMI-1640cul u emedium(SigmaR6504)
dissol edins e ilewa e wi h25mMHEPES,5.0%NaHCO3,
10% eshhumanA+se um(inac i a eda 56EC o 30min)
incuba edin5%O2,5%CO2and90%N2ana mosphe e.F esh
ed blood cells we e added wice a week.
In i o
an iplasmodial ac i i y by he SYBR G een I® me hod was
pe o medin heMala iaG oup,acco ding o he
me hodologydesc ibedbySmilks ein
e al
.19.Assayswe e
pe o medonFalcon®96-well la bo ompla es.Asuspension
o pa asi ized edbloodcellswi hahema oc i o 2.5%anda
pa asi emiao 1%we ep epa ed.Cul i a ionwi h ea men s
andchlo oquine(CQ)posi i econ olwe eincuba eda 37EC
o 48hin5%CO
2, 5% O2 and 90% o  N2 a mosphe e.
Subsequen ly, hecon en so eachwellwe e ans e ed o
G eine P oonedishesand hepa asi eswe elabeledwi ha
solu iono SYBR®G eenI2Xinlysisbu e .Thepla eswe e
incuba eda  oom empe a u ein heda k o onehou and
he ela i e luo escence uni s (RFU) we e ead on a
spec o luo ome e a 485nmexci a ionwa eleng hand
538nmemissionwa eleng h.T ea men swe ep epa ed oa
s ocksolu iono 10mgmLG1inpu eDMSOandsonica ed o
acili a edissolu ion, i s dilu ionwas1%andshowed obe
non- oxic o hepa asi e.F om hissolu ion,50µLwe e aken
andadjus ed o1000µLwi hcomple eRPMI-1640medium,
ob aining a inal concen a ion o  0.5 mg mLG1. Se en
concen a ions o  each ex ac  we e e alua ed in a ange
be ween100-1.56µgmLG1.Eachconcen a ionwase alua ed
in iplica eon hepla eand h eeindependen assayswe e
pe o med.TheCQcon olwase alua edina angebe ween
150-4.7nMand hecon olo Pe u ianquinaex ac (MeOH:
H2O;70:30)wase alua edin he angeo 0.01-10µgmLG1.
Da a om h ee ialswe eanalyzed o ind heinhibi o y
concen a ioninµgmLG1(IC50).Inhibi o yconcen a ions
50(IC50±SD)we ecalcula ed o eachcompound oma
non-linea logis ic eg essionmodel.Theda awe eanalyzed
andplo edusingG aphPadP ism4 o Macin osh e sion
4.0b which ou pu s he adjus men  alue ( ) (G aphPad
So wa e, San Diego, Cali o nia, USA). To classi y he
an iplasmodialac i i yo anex ac , heMala iaG oupo  he
Uni e si y o  An ioquia es ablished a consensus o  he
ex ac se alua ed:highlyac i e<5µgmLG1,p omising
6-15µgmLG1,mode a eac i i y16-30µgmLG1,lowac i i y
31-50µgmLG1andnon-ac i e>50µgmLG120-22.Toclassi y he
an iplasmodial ac i i y o  a compound, he Mala ia G oup
es ima ed ha acompoundisp omisingi  heIC50is<10µM23.
Cy o oxici y es ingandcalcula iono  heselec i i yindex
(SI):Theme hodo 3-(4,5-dime hyl hiazol-2-yl)-2,5-diphenyl
e azoliumb omide(MTT)acco ding oMosmann24,which
e ealscellula damagea  hemi ochond ialle el, oe alua e
hecy o oxicac i i yo  heex ac sandcompounds.U-937
andHUVECcellslineswe eused,whichwe emain ainedin
con inuouscul u esin heMala iaG oupLabo a o y.These
cells we e cul u ed a  37EC and 5% CO2inRPMImedium
supplemen edwi h10%inac i a edFe alBo ineSe um(FBS).
Themediachangeswe eassessede e y48ho acco ding o
60

Res.J.Med.Plan s,12(2):57-64,2018
pHchangeso  hemedium,wi h10mincen i uga iona
1000 pm and by eplacemen  wi h esh medium25. In
Neubaue 'schambe ,U-937cellswe ecoun edand
pla edina96-well la bo ompla e,200.000cellsmLG1in
RPMI1640mediumwi h10%FBS.Fo o he  heassay, he
HUVEC cells a e coun ed in  a  Neubaue   chambe   and
seededina96-well la bo ompla e2×105cellswellG1in
100µLo RPMI-1640mediumwi h10% e albo ine
se um.Theywe eincuba eda 37ECwi h5%CO2 o 72hin
hep esenceo eacho  he se enconcen a ionso each
ex ac and/o compoundande alua edina angebe ween
100-1.56 µg mLG1.Eachconcen a ionwase alua edin
iplica e in he dish and h ee independen  ials we e
pe o med. Subsequen ly, mi ochond ial dehyd ogenase
ac i i ywasmeasu edbyadding20µLwellG1o MTT oa
concen a iono 5mgmLG1andincuba ed o 3ha 37EC
unde 5%CO2.Todissol e he o medc ys als,100µLwellG1
o a50%solu iono isop opanoland10%SDSwe eadded
and he abso bance ead a  595 nm in an ELISA eade
(BioRad).Fo cy o oxici yassays, heposi i econ olwas he
cul u emediumand henega i econ olwasampho e icin
whichshowed oxici yin heU-937cellsandHUVEGcellline.
Da a om h eeindependen  ialswe eanalyzedusing he
G aphPadP ism5p og am o ind he oxicconcen a ion
inµgmLG1(CC50)usinganon-linea logis ic eg essionmodel.
The coe icien  o  a ia ion (% CV) was also es ima ed o
es ima e hedeg eeo dispe siono  heCC50ob ained om
he h eeindependen assays26.Toclassi y hecy o oxici yo
heex ac , heMala iaG oupo  heUni e si yo An ioquia
es ablished a consensus o  hesamplese alua ed:highly
oxic <10 µg mLG1, cy o oxic 10-40 µg mLG1, mode a ely
cy o oxic41-100µgmLG1andnocy o oxic>100µgmLG127.In
addi ion, heselec i i yindex(IS),whichindica esselec i i y
owa ds he pa asi e, was calcula ed as he ela ionship
be weency o oxicCC50ac i i yandan iplasmodialac i i y
IC50.Fo  heU-937celllinei wases ima ed ha IS alues
abo e2a econside edp omisingex ac s obee alua edin
heHUVECcellline(endo helialcellsisola ed om hehuman
umbilicalco d ein).Fo  heHUVECcellline,IS aluesabo e
5we econside edasp omisingex ac s.
S a is ical analysis: Measu emen s we e pe o med in
iplica eand he esul swe ep esen edas hemeanandi s
s anda dde ia ion(DS).Theda awe eanalyzedandplo ed
usingG aphPadP ism4 o Macin osh e sion4.0bwhich
ou pu s he adjus men  alue ( ) (G aphPad So wa e,San
Diego,Cali o nia,USA)andallcalcula ionswe epe o medin
hes a is icalp og amSTATGRAPHICSCENTURIUNXVI.
RESULTSANDDISCUSSION
The pe cen ages o  ex ac able ma e ial,
in i o
an iplasmodialac i i yinchlo oquine-sensi i e
Plasmodium
alcipa um
s ainNF-54,cy o oxici yinU-937p omonocy es,
HUVECendo helialcellsandde e mina iono  heselec i i y
indexo ex ac sandcompoundso 
P.piedecues anum
T el.
 and Yunck we e p esen ed in Table 1 and 2. They we e
classi ied acco ding o he an iplasmodial po en ial
es ablished by he Mala ia G oup o  he Uni e si y o
An ioquia.Thepe cen ageso ex ac ablema e ialwi h he
di e en  sol en s showed be e  yields o  he
dichlo ome haneex ac so  hes emsandlea esmix u eo
he
P.piedecues anum
(PPD)specieswi h1.75%,howe e no
associa ion was ound be ween ex ac ion yields and he
biological es s ha we epe o med.Fo allex ac so  he
species
P. piedecues anum
 T el. and Yunck. ound an
adequa e concen a ion- esponse ela ionship wi h
s a is icallysigni ican co ela ioncoe icien s(R²) o all he
samplese alua ed.Thechlo oquineposi i econ olhada
mean alueo IC50=26.1±5.4nM and hecon olo  he
Pe u ian quinine ex ac  (MeOH: H2O; 70:30) p esen ed a
mean alueo IC50=0.32±0.16µgmLG1.
Table1:Cy o oxici yandan iplasmodialac i i yo  heex ac so di e en pola i ieso 
P.piedecues anum
T el.andYunck
Pe o manceo
ex ac ion(go IC50(µgmLG1)X±SD* CC50(µgmLG1)X±SD CC50(µgmLG1)X±SD
ex ac /go d y ---------------------------------------- -------------------------- ----------------------------
Code Ex ac bype cola ion,lea esands ems plan ma e ial)(%) S aino 
P. alcipa um
NF-54** CelllineU-937 IS*** CelllineHUVEC***** IS***
PPH Pe oleume he ,25ECZP*:5mm10days 0.7 >50 24.9±4.1 ND NE ND
PPD Dichlo ome hane,25ECZP*:5mm10days 1.7 17.9±2.02 37.6±1.7 2.1 101.4±2.7 5.7
PPAE E hylace a e,25ECZP*:5mm10days 1.1 19.5±1.8 15.4±2.6 0.8 80.7±1.6 4.1
PPM Me hanol,25ECZP*:5mm10days 1.6 >50 42.3±0.23 ND NE ND
PPE E hanol25ECZP*:5mm24h 0.9 25.2±0.3 12.4±0.33 0.5 NE NE
*ZP:Pa iclesize,**Da ao asingle eplica,***X(A e age)+SD(S anda dde ia ion),****Chlo oquineposi i econ olIC50=26.10±5.37nM,pe u ianmachineex ac
0.32±0.16µgmLG1,*****Selec i i yindex(IS)=CC50(µgmLG1)/IC50(µgmLG1),******ND:No de e mined alueo IC50>50µgmLG1,Classi ica iono an iplasmodial
ac i i y:highlyac i e<5µgmLG1,p omising6-15µgmLG1,mode a eac i i y16-30µgmLG1,lowac i i y31-50µgmLG1andnon-ac i eac i i y>50µgmLG1.
Classi ica iono cy o oxicac i i y:highly oxic<10µgmLG1,cy o oxic10-40µgmLG1,mode a elycy o oxic41-100µgmLG1andnon-cy o oxic>100µgmLG1
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120
100
80
60
40
20
0
Inhibi o y concen a ion 50 (IC y CC µg mL )
50 50
G
1
IC
50
CC U-937
50
CC HUVEG
50
PPHEx H
PPHTEx D
PPHTEx A
PPHTEx M
PPHTEx E
Ex ac s o di e en pola i y (PP)
P. pidecues anum
*
+
Table2:An iplasmodialac i i yo isola edcompoundso 
P.piedecues anum
IC50(µgmLG1)X±SD* CC50(µgmLG1)X±SD
---------------------------------------- ---------------------------
Code S uc u e S aino 
P. alcipa um
NF-54** CelllineHUVEC IS***
1 7.3±0.4o 25.7µM >100 13.7
HCO
3
OH
O
OOH
2 >50 >100 ND
HCO
3
HCO
3
O
O
OH
OH
3 >50 60.57 ND
HCO
3
HCO
3
O
O
OH
4 >50 >100 ND
HCO
3
HCO
3
O
O
OH
5 >50 >100 ND
HCO
3
O
O
OH
6 >50 >100 ND
HCO
3
HCO
3
O
O
OH
B
*X(A e age)+SD(S anda dde ia ion),**Chlo oquineposi i econ olIC50=0.008±2.78µgmLG1o IC50=26.1±5.4nM,***Selec i i yindex(IS)=CC50(µgmLG1)/IC50
(µgmLG1)IS>2con i mse icacyandsa e y,ND:No de e mined.Thede ini iono  hean iplasmodialac i i yusedwas:IC50<5µgmLG1-s ongac i i y,6-15µgmLG1-
mode a eac i i y,16-30µgmLG1-sligh lyac i eandIC50>30µgmLG1-inac i e
Fig.1: Compa ison  o  he an iplasmodial andcy o oxic
ac i i yo ex ac so di e en pola i yo
P.piedecues anum
Thebiologicalac i i y ound o  heex ac so  hespecies
P.piedecues anum
T el.AndYunckshow ha  hepe oleum
e he  ex ac  (PPH) wi h non-pola  componen s and he
me hanolic ex ac  (PPM) wi h pola  componen s did no
p esen an iplasmodialac i i y(IC50>50µgmLG1).Howe e ,
ex ac s o  dichlo ome hane and e hyl ace a e showed
mode a ean iplasmodialac i i ywi hIC50=17.9µgmLG1;
ISHUVEG=2.09andIC50=19.5µgmLG1;ISHUVEG=0.79,e en
p esen ingbe e pha macologicalac i i y han hee hanolic
ex ac wi hIC50=25.2µgmLG1;ISHUVEG=0.49(Fig.1).I canbe
in e ed ha  hee ec o  heseex ac sisdue ocomponen s
wi hmode a elypola andnon- oxiccha ac e is ics,since o
heex ac o dichlo ome hane,whichwas hemos ac i e
andselec i e,aselec i i yindex>2wasp esen ed,indica ing
hespeci ici yo ex ac  owa ds hepa asi e.The ac  ha  he
cy o oxici yo  hisex ac willinc easeCC50=37.63µgmLG1
wi h espec  o he o he  ex ac s indica es ha  he
componen sp esen in hisex ac a epoo lycy o oxic.In
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addi ion, when hese ex ac s we e e alua ed in HUVEG
me abolicallyac i ecellsanin e es ingISwas ound,sincei
wasshown ha  heex ac sdidno a ec  his ypeo
cells,since hei cy o oxicconcen a ionswe eCC50=101.35;
ISHUVEG=5.65andCC50=80.7;ISHUVEG=4.14, o  heex ac so
dichlo ome haneande hylace a e espec i ely.
Fo  species o  Pipe  genus, p omising ac i i y agains
Plasmodium in
P.capense
L.specieshasbeen
demons a ed.(Pipe aceae)wi hanIC50=7.0µgmLG1ac i i y
in
P.hos mannianum
s ain(W2)chlo oquine esis an 28
wi h an IC50=8.0µgmLG1,
P.umbella um
wi h70%
inhibi iona 40µgmLG1and
P.sa men osum
wi han
IC50=0.05µgmLG129.
In i o
an imala ialac i i yo  he
Pipe be le
  lea   me hanolic  ex ac   e alua ed in
Plasmodium be ghei
 (NK65) in ec ed mice has been
demons a edo e a angeo concen a ions
(50-400mgkgG1)30.Theac i i yo (-)-me hylende a inwas
con i med
in i o
 in mice in ec ed wi h
P. inckei pe e i
,
showingan80% educ ioninpa asi emiaa adoseo
20mgkgG1dayG131.I is he i s  ime ha  heme aboli es
ha e been epo ed 5,8-Hyd oxy-7-me hoxy la one(1),6,7-
dime hoxy-5,8-dihyd oxy la one(2), 6,7-dime hoxy-5-
hyd oxy la one (moslo la one) (3), 5,6-dihyd oxy-7-
me hoxy la one(negle ein)(4),5-hyd oxy-7-me hoxy la one
(5) om
P. piedecues anum
 species and a b omina ed
de i a i e om (6) named 6,8 b omo-5-hyd oxy-7-
me hoxy la one(7)in hespecies
P.piedecues anum
.The
compound(1)was heonlyone ha p esen edan iplasmodial
ac i i ywi hanIC50=7.325µgmLG1(25.69µM);ISHUVEC=13.65.
The b omina ed de i a i e o  (5) also did no  exhibi
an iplasmodialac i i y,indica ing ha  o  hiscompound he
p esence o  halogena ed g oups does no  signi ican ly
in luence he he apeu ic esponse. O he  species o  his
genusha e epo ed a ious la onoidswi han iplasmodial
ac i i y. The compound 6-p enyl-3'-me hoxye hyliode hiol
showednoac i i yon he
P. alcipa um
s ain.Analysiso  he
s uc u e-ac i i y a ioshows ha  hep esenceo adjacen
me hoxy and hyd oxyl g oups o  he absence o  adjacen
hyd oxylg oupscouldcon ibu e o heinac i i yo  he
6-p enyl-3'-me hoxye hyliode hiol compound and he
subs i uen sin he la anonebackboneclea lyin luence he
an iplasmodial ac i i y32. The an iplasmodial ac i i y o
la onoids is a ousing a g ea  in e es  in he chemical
medicine,since heya eo ien ings udieso s uc u e ela ion
SARo chalconesandde i a i eso  la onoidswi hdi e se
subs i uen sin ingB,AandC.Chalconesa emo eselec i e
hanme hoxy la ones,chalconede i a i ecompoundsha e
amo eselec i eac i i y(6.7-16.9µM)o e 
P. alcipa um
(W2)
han la onoid de i a i es (5-33 µM)32; me hoxy la ones
p esen  hebes ac i i ies,e en hese esul scomplemen  he
alidi yo  he esul sob ainedin hiswo k o  heisola ed
compound5,8-hyd oxy-7-me hoxy la one(1).
CONCLUSIONANDRECOMMENDATION
Thean iplasmodialandcy o oxicac i i yo  hespecies
P.piedecues anum
was epo ed o  he i s  imeinwhich
hemode a elyac i eex ac swe e hoseo dichlo ome hane
ande hylace a ewi h hebes e ec o an iplasmodial
andcy o oxicac i i ywi hIC50=17.93µgmLG1;IS=2.093and
IC50 = 19.5 µg mLG1; IS = 0.791, espec i ely. The
an iplasmodial e ec  o   hese  ex ac s  is  due  o
componen s wi h mild  pola   cha ac e is ics  and  li le
oxici y,whichmo i a escon inuingwi h hes udyo  he
SARs uc u e-ac i i y ela ionshipo  la onoidswi h a ious
subs i uen sin ingB,AandC.
ACKNOWLEDGMENTS
The au ho s a e g a e ul o  he inancial  suppo
p o idedby heColombianMinis yo Ag icul u e
(No.009-2007-V7552-38-07),Colciencias h ough heJoséde
Caldas anchisesand heUni e si yo An ioquia.
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