1. — 301 —
Trypanocidal Constituents in Plants 1. Evaluation of Some Mexican
Piants for Their Trypanocidal Activity and Active Constituents in Guaco,
Roots of Aristolochia taliscana
Fumiko A H E , * " Shinya NAGAFUJI,"Tatsuo YAMAUCHI," HikaruOKABE," Jun MAKI.'' Hiroo HIGO,'
Hiroshige A K A M A N E / Abigail A C U I L A R / Manuel JIMÉNEZ-ESTRADA/ and Ricardo REYES-CHILPA'
" FMuIry of Pharmaceiiiical Sciemes. Fukuoka Vnivemty: ""School of Medicine. Fukuoka Univeríiiy: H-IV-/ Nanakuma.
Jonan-ku. Fukuoka HI4-OIH0. Jopan: "SchooloJMedicine. Kiiasuiu Vñiverxiiy; I-15~¡ Kilasaio. Sagamihara 22S-H5f5.
Jopan: ' Gniduate Scfiool oj Medical Sciences. Kyushu Universily: 3 l-¡ Maidashi. Higashi-ku. Fukuoka Iil2
Japan:''Herburium. Mexican /nsiitufe Jór Social Securin: Salional Medical Crnter: Cuauhiemoc 3ÍÜ. Ü672S. México D.
F. México: and' Insiituie of Chemisiry. Sational Univenify of México: Ciudad Umversifaria. 04510. México DE. México.
Received Apríl 22. 2002: acccpted June 12. 2002: publishcd oniinc June 17. 2002
Crudc fitricli of MCXÍCIB medicinal plaati were Kreencd for trypiaocldal activity agalnst Trypanosoma
cruzi, which li (be etiologtcal agcat for Chagas' discase, onc oí tke mosl tcrious protozoan dlscases In Latin
America. Therr were 43 klnds of meihanoNc and other organlc estracts from 39 plants whkh wrrc eiamlned by
thc preljmlnary Kreening test to tee ImmofallizatiQn of cplraastlgotes of T. cruzi in vitro. EJghlcen of them showed
•ciivit> al tkc conccniratlon of 2 mg/ml afttr incubatlon for 2 K. whilc 13 showed acilvit>' al the concentration of
1 mg/ml alter incubatlon for 48 fe. A m o n g them, the M e O H extract of rools of Aristolochia tatiscana (ArHlolochi-accac),
locally known as "Guaco," immobllúed all ihe cpinastigotei even at lowtr conceatrailon of 0.5 mg/ml
(48 ht. In ordcr to Identlfy principal corapounds for thls activity. the M e O H eiiraci of Guaco was subjecicd to
Moassay-guided fractionalton. From the active fractloos, foor neoUgnans. euporoalcnoid-7 (I), llcarin A (2>, cu-pomalcnoid-
1 (5) and Ikarín B (6), and t*vo tignani, austroballlgnan-T (3) and íragransin E, (4) wer« Isolated.
Compounds I—4 immobllizcd all the cplmaitigotes al the mínimum concentration otlS—75/^/ml after incuba-tion
for 48 h, while compounds 5 and 6 were Inactlve. Correspondtng conccniratlon of gossypol, bcrbcrinc chlo-rlde
and harmlnc was 280 fi%/ml, 300 /ig/ml and >500 >i^mL respectivcly.
Key wordi trypuiocidaJ «ctivity. Try-pamotoma enai; AristotiKhia udiscaiia, Chagss' diseasc: neolignan; liyiuui
Protozoan Trypanosoma cruzi ís the etiologica) agent of
Chagas' dísease (American trypanosomiasis), which affects
16—18 million people in Latin Amenca and is rcsponsiblc
for thc death of more than 45(X)0 paiients per ycar." Il is
transmitted to humans by tnatomine bugs or through blood
transfusión. During its life cycie. T. cruzi diffeimtiates inio
three stages. namely cpimastigote in the insect gut. trypo-mastigote.
an infeciious form in the blood stream, and
amastigote. an intraceitular form.
Medication for Chagas' disease is usually efTeciive when
gtven during the acute slage of infection. Once the disease
has progrcssed to iater stages, no medication has been proven
to be effeciive- Moreovcr, synthetic drugs. such as nifurtimox
and benznidazole. have severe side cffects.^-'' There rises an
urgent need lo develop new drugs. in order to seek new
chemotherapcutic agents from natural resources. wc slarted a
survey of trypanocidal constituents in Mexican plants. Inves-tigalion
on a trypanocidal constiluent in Piquería innervia,
one of Mexican plants. was already performed by Castro ef
al** A S a preliminary screening test, we examined crude ex-tracts
of Mexican medicinal plants for trypanocidal activity
againsi epimasligotes of T. cruzi in vitro as previously re­poned
in the case of plants in Guatemala^' Among them the
MeOH extract of rools of Aristolochia taliscana (Aris-lolochtaceae)
showed trypanocidal activity. A. taliscana and
some other species of Aristolochia are locatly called
"Guaco" and uscd as remedies for diarrhea, snake hites, and
dermatológica! affeclions.*" Chemical investigation of A. tal­iscana
has been previously accomplished by Enriquez et al
and four ncolignans have been isolated.^'
In this paper we describe thc results of preliminary screen­ing
tests for trypanocidal activity in some Mexican plants.
and identifícation of the active constituents in Guaco. A tal­iscana,
onc of the plani materials which showed activity.
MATERIALS AND METHODS
Piani Materials and Preparation of Their Extraéis
Plant materials including A. taliscana were mainly purchased
at Sonora medicinal plant market in México City and col-lected
in the fieids. Identifícation of thc plants was done by
M. E. López-Villafranco of National University of México
(Iztacala) and A. Aguilar, one ofthe co-authors. The voucher
specimens were deposited m the Herbanums IZTA and
IMSSM of National University of México (Campus Iztacala)
and Mexican Institute for Social Security. respcctively. The
plants examined are listed in Table I. Oned and powdercd
materials were cxtracted wUh McOH. MeOH-CHjClj or acc­ione
at room tcmpcrature ovemight, In the case of frcsh ma­terials,
chopped matenals were soaked in MeOH and fittered.
The residuc was extracled again with MeOH. The soKcnt
was concentraied in mcMo lo give each extract,
Cultivation of T. cruzi The strain of T cruzi used in this
study was H6 (international code: MHOM/GT/V5'SMt-06).
which was originatly collected from a patient of Chagas' dis­case
in Guatemala by Dr. T. Yanagi of Nagasaki Univetíity.
Dr. C. Monroy and Dr. V. Malta of San Carlos University in
Guatemala, and H. Higo, onc of the co-authors. The epi-mastigoles
of T. cruzi have been culturcd in tiver infiision-tryptosc
(LIT) médium as described by Baum.*' Hemin was
replaccd by hemc^lobin.
Reagcnts Tryptose and líver infusión broth w m ob>
• To whom corrcspondcncc should be Mldmscd c-matl: abcfumi'á^ fukuolca-u.K.jp ' 2002 Pharmaccutical Societv ot Jifur
( 1 )

2. — 302 —
[•upom«1»nw*-7) y llicwin*)
(•tjQomoMooMl-i)
-CHj.
3 (tuatrobMlignan-T) 4 (tr»gi«n«in e, I
i-) MthyIpiwwioW*
Fig. 1. Isolaied Compounds from Roooof'l laliscanu
GoMypol BortMrlna chiodf
lained from Difco. fetal bovine senmi from GIBCO and
hemoglobin from Japan Biotest Institute. Gossypol and
harmine were purchased frt>m Sigma Chemical Company,
and berberíne chioride (n-hydrate) from Tokyo Chemical In­dustries
Co. Ltd.
Trypanocidal Ansy Preliminary Screening: Each ex­tract
was dissolved in dimethyl sulfoxíde (DMSO) ñrst and
then diluted with LIT médium to get certain concentration.
The final DMSO concentration was less than 1%. 1% DMSO
solution itself caused no affcction on motion of epimastig-otes.
Under condition I, the fína) concentration of each ex­tract
was 2 mg/ml and incubatlon time was 2 h. Under condi-tion
2, the final concentration was 1 mg/ml and incubation
time was 48 h. Each 50;J1 of sample solution and cell sus­pensión
(ca. 2X10^ epimastigotes/ml) was placed in a 96-
wel) micro píate in duplícate and incubated at 26 °C. The
control was free from samples. The motion of cpimastigotes
both in the sample well and in the control well was observed
under inverted lighi microscópe (xlOO). Each test was run
twice. The results are shown in Table 1, Tlie mark (+) means
that all the epimastigotes became tmmobilized, while the
mark (±) means 80—90% of the whole epimastigotes be­came
immobiüzed, The mark ( - ) means that more than 50%
of the epimastigotes kept mobility.
Estimation of Trypanocidal Activity: Sample solutions in
different concentration were trealed as mentioned above. The
activity is shown by MC,,^ valué, which was defined as the
mínimum concentration at which all the epimastigotes be-come
immobilized after 48 h-incubalion al 26 °C.
Extnction and IsoJatloa of thc Active Conititueiits
from thc Roots of Aristolochia taüscana Dríed and pow-dered
roots (I42g) of A. taliscana were cxtracted with
MeOH (500ml) under reflux for I h and fiitcrcd. Further ex-traclion
for 30min was done twice. The filtratcs were com-bined
concentrated and dríed in vacuo to givc a dark brown
residue (19.4g). The MC,oo valué of the MeOH extract was
0.5 mg/ml. Thc residue was suspended in 60% MeOH and
centrifuged. The precipitates were cxtracted with MeOH. and
then with AcOEt. Thc supematant of 60% MeOH was passed
through a column of slyrene polymer, Diaion HP-20, and the
column was washed with 60% MeOH. The MeOH solution
was passed through the same column and the column was
washed with MeOH, The AcOEt solution was treated in the
same way. Thc 60% MeOH eluate was concentrated in vacuo
10 remove MeOH and the aqucous solution was passed
through a new Diaion HP-20 column. The column was eluted
with HjO and then 60% MeOH. Each eluate was concen­trated
and dríed in vacuo to obtain brtywn resin: HjO eluate
(fr I, lO.I g); 60% MeOH eluate (fr, 2, 1.4g); MeOH eluate
(fr, 3, 5,2 g); AcOEt eluate (fr, 4, 2.3 g). MC,oo valúes of frs.
1—4 are >IOOO^g/ml. >1000;ig/m!. 60pg/ml, and 400
^g/ml, respectively, Fractions 3 and 4 were chromatographed
with silica gel (hexane-AcOEt), Sephadex LH-20 (CHCIj),
and ODS (YMC gel) (70% MeOH) column, successively. to
afford cupomatenoid-7" (I, 826mg), licarin-A"*' (2, 324
mg), mixture of 3 and 4 (230mg), cupomatcnoid-1 "* (5, 109
mg), and licann B'^' (6, 368mg). Austrobailignan-?'*' (3,
139mg) and fragransin E,'*' (4, 35 mg) were separated by
preparative HPLC (ODS. KC-Pack. 60% MeOH). Identifica­tion
of compounds 1—6 was performed by analyses of MS
atxl NMR spectra as well as the comparison of physícal data
with those reponed, MC|Q Q valúes of compounds I—6 were
estimated as descnbed above and shown in Table 2.
RESULTS AND DISCUSSION
Many trypanocidal constituents from natura! resources
have been isolated and were fírst reviewed by Scpúlveda-
Boza and Casseis in 1996.'** In this rcvicw, various kinds of
ixatural compounds were reponed such as hydroquinones,
naphthoqui nones, diterpenes and many types of alkaloids. Il
is diflicult to compare activity of each compound due to the
diversity of strains, stages of its life cycle, and experimental
conditions applied, Since Schlemper et al. mentioned the
positive correlation betwcen activity against epimastigotes in
vitro and activity against trypomastigote in vivo,'*' we used
epimastigotes to estímate trypanocidal activity. The method
of assay by Hocquemillar et al. was modifíed for estimation
of trypanocidal activity in vitro.^^^
For preliminary screening tests, we chose plants and
herbal medicines traditionally used mainly for antiparasitic
purposes in México'*' and Guatemala,"' Thc list of crude cx-tracts
is shown in Table 1 with scicntifíc ñames, local ñames
and pans examined. It is comprísed of 20 families and 37
( 2 )

3. Trypanocidal Constituents in Plants 1. Evaluation of Son» Mexican Plants for Their Trypaoocldat
Actiity and .Active Constituents in Guaco. Rools oí Arisioioch'm laliscaim (Fumiko Abe. et aJ. í — 3 0 3 -
T W c 1. Littof Plañís Euminedand Their Tryputocidai Activity'(E pimattiifoiet. in l'iTw)
Family Sckwific ñame Local ñame Pan iSolvent)" llf
Annonaceae Amutna nrliivlatu Anona LT(M| + +
Aaniina miiriiaiu Guanábana S(M) +
AnstolvchMccae AnMii/ttthni latiMami Guaco R(M| • +
Buncraccac Buneni ximaruha Palo mulato LT (MI - F|M) -
Cecropuceae Crti^a ohiu.lh>lia Guarumbo L(M) + - Chenopodiaccac CMirmi/KMtium gnnviiírn. t-pa/uie de zomllo C(M)
ChvmiiKHhim <iiuhnisi/ndr Epatóle morado C(M) - - Compon tae ArirmitHi liidtntcíaiui VK. mr.TÍcaEsiiuaiA aie L(M) - BlJtn^ iitlorula 1o«»te blanco G(M) + t
CiKiubtuccse Klaximiminia ínmirue Guateque RlM> • "- - ElacocatpaccM Mumuifiw calutwu Capulín rojo L(M) , + - F(M) — - Euphorbiaccac Cnuim Jraco Sant¡re de ipwdo L(M) - Hitni ptiliamlni Haba o Habílla smt - - - CuRí/rne Cuiophy lluni hru.silien.ie'' Ban LÍA) +
Ca¡ophllum hrasilienst" Ban L(M) Clusiii salvinii Lobo de ligre H M + C t - - Cliaia gtiairmulensis Lobo de tigre L(M) -Can mili iniermedia Limoncillo L { M + C ) - -S4ammea amernanu Zapóle Domingo P(A» + -
fLimia bacci/era Vismia L(M) —
¿auncew Prnta umehcana Aguacate SíMí -
Leguminouc BrDHftnianUi potlalvrioide% Hieiba de la Vfbon R(M1 Eviciihunia poltuocli ia Palo Dulce StMl - - CHlairtmii'aidlaiax ysleupmtu mhr asilelo CPoacluoi diee Bnul LS((MM() -+ --
Lomhuí'urpus uniloUoUtium L(M) - - LonchtKarpia imxacensis R(M) — Senna hirsuta Yccapaht2in LT(M) Zoniia ihi-miMla -
Hierba de la víbora L(M) + •
Marvaceae Malvamcus arhorwus Azocopacle L(M) - - MyrtKcae PsiJiwn ¡guajava Guayatn L(M| - SIM) — —
Pipenceae Piperíp L(M) + + hiypodiaceae Piper auritum Acuyo L(M) - - PhUhodium aureum Lengua de ciervo G(M) - < R(M) - Rubiaceac Hamelia patens Balletilla LT(M) - - Sapotaccae Pnutena iopoia Mamey S(M) - Unicaceae Vrtica dioicu Ortiga LT(M) Verbenaccac Almila mptnlla Tí cedrón L ( M ) - - Lippia dulcis Hierba duke L(M) —- -—
ul Thc mart I +1 mcaiH all (he cpiRiaitifMe* »«T« immabiliicd The mark (11 mean BO -90% of the whole epimattifoict ««re immobiliNd l>) IT lenet and t*ifi. R
Riots. F ftuñL L leavei.C trawMl iwu. S Mcnn. P pecH<>f fhiiiv M McOH. A i M I ^ r) Acinity ai In^riil (2li). rf) Acoviiy at I i^'ml (4tb). c} CeA-Icctnj
ti Sanu Vtanha. Vcncnu f} C'oJIecud at La« TuxtlM. Wcracnu.
Species. Eighteen out of 4 3 extracts showed trypanocidal ac­tivity
so far at 2mg''ml. while 13 showed activity even at 1
mg/ml We slarted the chemical investigation of these active
plant matenals.
The MeOH extraéis of roots of A. taliscana immobilized
all the epimastigotes at O.S mg/ml. When the MeOH extract
was separated into 4 fractions (frs. I —4) by a Diaion HP-20
column, activity was observed in less polar fraction 3 eluted
with MeOH and fraction 4 eluted with AcOEt. Fractions 3
and 4 were combined and subjected to vanous kinds of chro-maiographies
lo afford four neolignans, eupomatenoid-7 (I),
licann A (2), eupomatenoid-1 (5) and ticann B (6), and two
Itgnans, austrobailignan-7 (3) and fragransm E, (4). Al-though
these neolignans were already isolated from Guaco
by Enriquez el al..^* this is the first isolation of lignans 3 and
4 from Guaco. MC,,H) valúes of these compounds are listed in
Table 2. Trypanocidal natural compounds, gossypol.^'
Table 2 MC,^ Valúes of Compounds I—«. Gouypol, Berbenne Chio­ride.
and Harmine againsi Epimastigotes of T cruzi In Viim
MC|op
CompouiKU
1 2S
s 40 s 123 75 4 219 SO 146 >IO00
« >IODO
Gossypol 280 540
Bcrbérinc Cl
Harmine >500
( 3 )

4. — 304 —
berbcrine chioride^" and harmine^'* were aiso estimated and
MC,oo valúes of them are listed in Table 2. Among six com­pounds
tsolated from Guaco. 1—4 exhibited higher activity
than the above mentioned three compounds. Comparisons of
1 with 5, and 2 with 6 suggest that the loss of hydroxyl group
reduces activity. The differences of activity between 1 and 2,
and 3 and 4 are nol negligible. It suggests that stenc struc-tures
might influence on activity. Judging from the yieid and
MC|oo valué, trypanocidal activity of Guaco is mainly due to
I (eupoinatenoid-7).
Recently Bastos et ai ha isolated seven lignans from
Zanthoxylum naranjillo (Rutaceae) and revealed that ( - ) -
methyipulviatolide is hi^ly active against trypomastigotes
of the two strains of T. cruzi, the Bolivia and Y."' As for try­panocidal
lignans, lignans from Guaco are the second in-stance.
Chemical investigation of the other active plant extracts
and bioassay against trypomastigotes in vitro are now under
progress.
Ackoowledgements The authors are gratcfiil to Dr. M.
A. Martínez Alfaro for valuabte suggestton on the medicinal
plants in México. The authors are also grateful to Dr. M. E.
López-Villafranco, Natioiial University of México. Campus
Iztacala, for the auihentication of the plant specimens. We
thank Ms. Y Iwase and Mr. H. Hanazono for NMR and MS
measurements. This work was supported in part by the fund
(No. 001001) from the Central Research Institute of Fukuoka
University: 2000-2001.
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lOOH), Gcntian violei as a positive control.
( 4 )