As a preliminary screening test for the trypanocidal activ-ity against epimastigotes of Trypanosoma cruzi in vitro, weexamined some crude extracts of Mexican medicinal plants.2)

T. cruzi is the etiological agent for Chagas’ disease (Ameri-can trypanosomiasis), one of the most serious protozoan dis-eases in Latin America. Then we started on identification ofthe principal compounds of the active extracts seeking newchemotherapeutic agents for Chagas’ disease. Lignans2) fromGuaco (roots of Aristolochia taliscana) and xanthones3) fromBari (stems of Calophyllum brasiliense) were isolated as ac-tive components against epimastigotes in vitro.

T. cruzi exhibits three developmental stages: epimastigotein the insect gut; trypomastigote, an infectious form in themammalian blood stream; and an amastigote, a proliferativeform in mammalian cells. In this paper we describe the results of continuous preliminary screening tests for try-panocidal activity against trypomastigotes as well as againstepimastigotes in some Mexican plants.

The edible fruit of Persea americana MILL. (Lauraceae),avocado, is grown throughout the tropics worldwides. TheMeOH extract of seeds of Persea americana also showedmoderate activity in the screening test. Herein we also de-scribe the identification of the active constituents in seeds ofP. americana.

MATERIALS AND METHODS

Plant Materials and Preparation of Their ExtractsPlant materials were mainly purchased at a medicinal plantmarket in Guadalajara, Jalisco, and collected in the fields.Identification of the plants was done by M. A. Martinez-Al-faro, one of the co-authors. The voucher specimens were deposited in the Herbarium of Jardín Botánico del Institutode Biologia, Universidad Nacional Autonoma de Mexico.

The plants examined are listed in Table 1 including those referred to in ref. 2. Dried and powdered materials were extracted with MeOH or MeOH–CH2Cl2 at room tempera-ture overnight. In the case of fresh materials, chopped mate-rials were soaked in MeOH and filtered. The residue was extracted again with MeOH. The solvent was concentrated invacuo to give each extract.

Cultivation of T. cruzi The strain of T. cruzi used in thisstudy and the cultivation method of epimastigotes were thesame as those described in the previous paper.2) The trypo-mastigotes were harvested from the culture supernatant ofrhesus monkey kidney (LLC-MK2) cells infected with T.cruzi as described in the previous paper.4,5) LLC-MK-2 cellswere maintained in DMEM containing 10% FBS at 37 °Cunder 5% CO2. After inoculation of trypomastigotes intoLLC-MK2 cells, the medium was changed to DMEM con-taining 10% NCS.

Instruments and Reagents The instruments and reagentsused in this study are the same as those described in the pre-ceding paper.1)

Trypanocidal Assay The detailed assay procedure againstepimastigotes was described in the previous paper.2) The ac-tivity was expressed as the MC100 value (mg/ml or mM),which was defined as the minimum concentration at whichall the epimastigotes were terminated after incubation for24 h at 26 °C. Assay procedure against trypomastigotes wasdescribed in the previous paper.3,4) The activity was ex-pressed as the MC100 value as in the case of epimastigotesafter 24-incubation at 37 °C under 5% CO2. IC50 values wereestimated using Cell Counting Kit-8 as described in the pre-ceding paper.1)

Assay of Cytotoxicity The cytotoxicity of isolated com-pounds against human uterine carcinoma (HeLa) cells wasexamined using a modified MTT assay.6)

∗ To whom correspondence should be addressed. e-mail: abefumi@fukuoka-u.ac.jp © 2005 Pharmaceutical Society of Japan

Trypanocidal Constituents in Plants 5.1) Evaluation of Some MexicanPlants for Their Trypanocidal Activity and Active Constituents in theSeeds of Persea americana

Fumiko ABE,*,a Shinya NAGAFUJI,a Masafumi OKAWA,a Junei KINJO,a Hiroshige AKAHANE,b

Tetsuya OGURA,c Miguel Angel MARTINEZ-ALFARO,d and Ricardo REYES-CHILPAe

a Faculty of Pharmaceutical Sciences, Fukuoka University; b School of Medicine, Fukuoka University; 8–19–1 Nanakuma,Jonan-ku, Fukuoka 814–0180, Japan: c Department of Chemistry, CEN, Universidad Autonoma de Guadalajara; Av. PartiaNo. 1201, Guadalajara, Jalisco, Mexico: d Jardín Botánico del Instituto de Biologia, Ciudad Universitaria; and e Institutode Quimica, Universidad Nacional Autonoma de Mexico, Ciudad Universitaria; 04510, Mexico D.F., Mexico.Received February 15, 2005; accepted April 7, 2005

Crude extracts of Mexican medicinal plants were screened for trypanocidal activity against Trypanosomacruzi, which is the etiological agent for Chagas’ disease, one of the most serious protozoan diseases in LatinAmerica. There were 71 kinds of methanolic and other organic extracts from 65 plants, which were newly exam-ined by a preliminary screening test to observe immobilization of epimastigotes and trypomastigotes of T. cruzi invitro. The MeOH extract of seeds of Persea americana (avocado) showed moderate activity against epimastigotes.In order to identify the principal compounds for the activity, the MeOH extract was subjected to bioassay-guidedfractionation. From the active fractions, six 1,2,4-trihydroxyheptadecane derivatives and two 1,2,4-trihydroxy-nonadecane derivatives including a new one were isolated. These compounds showed moderate activity againstepimastigotes and trypomastigotes.

Key words trypanocidal activity; Trypanosoma cruzi; Persea americana; Chagas’ disease; 1,2,4-trihydroxyheptadecane deriva-tive

1314 Notes Biol. Pharm. Bull. 28(7) 1314—1317 (2005) Vol. 28, No. 7

Extraction and Isolation of the Active Constituentsfrom the Seeds of P. americana Seeds of P. americanawere gathered from imported avocados. Dried and powderedseeds (340 g) were extracted with MeOH (1 l) under refluxfor 1 h and filtered. Further extraction for 30 min was donetwice. The filtrates were combined, concentrated and dried in vacuo to give a dark brown residue (49.2 g) (MC100 :500 mg/ml). The residue was suspended in 60% MeOH andcentrifuged. The precipitates were extracted with MeOH, andthen with AcOEt. The supernatant of 60% MeOH was passedthrough a column of styrene polymer, Diaion HP-20, and thecolumn was washed with 60% MeOH to give fraction 1. TheMeOH soluble part of the precipitates was passed throughthe same column and the column was washed again withMeOH to give fraction 2. The AcOEt soluble part of the pre-cipitates was treated in the same way to give fraction 3. Eacheluate was concentrated and dried in vacuo to obtain a brownresin: 60% MeOH eluate (fr. 1, 38.0 g); MeOH eluate (fr. 2,6.5 g); AcOEt eluate (fr. 3, 4.2 g). MC100 values (mg/ml)against epimastigotes of frs. 1—3 are �1000, 250, and 1000,respectively. Fraction 2 was chromatographed with silica gel(CHCl3–MeOH, 20 : 1—4 : 1), Sephadex LH-20 (CHCl3),and ODS (YMC gel) (80% MeOH) column, successively, to afford 4-acetoxy-1,2-dihydroxyheptadec-16-ene7) (1, 107 mg),1-acetoxy-2,4-dihydroxyheptadec-16-ene8) (2, 60 mg), 1,2,4-trihydroxyheptadec-16-ene8—11) (3, 221 mg), 4-acetoxy-1,2-dihydroxyheptadec-16-yne12) (4, 32 mg), 1-acetoxy-2,4-dihy-droxyheptadec-16-yne10) (5, 4 mg), 1,2,4-trihydroxyheptadec-16-yne10,11) (6, 217 mg), 1,2,4-trihydroxynonadecane11) (7, 4mg), and 8 (3 mg). Identification of compounds 1—7 wasperformed by analyses of MS and NMR spectra as well asthe comparison of physical data with those reported. MC100

and IC50 values of compounds 1—8 were estimated as de-scribed above and shown in Table 2.

Compound 8: A solid, [a]D24 �8.6° (c�0.25, CHCl3), high-

resolution (HR)-FAB-MS m/z: 337.2721 ([M�Na]�, Calcdfor C19H38NaO3: 337.2719). 1H-NMR (500 MHz, CD3OD) d : 0.90 (3 H, t, J�7 Hz, H-19), 1.50 (1 H, dt, J�14, 8 Hz, H-3a), 1.68 (1 H, dt, J�14, 4 Hz, H-3b), 2.01 (2 H, m, H-8),2.17 (2 H, m, H-5), 3.44 (1 H, dd, J�11, 6 Hz, H-1a), 3.49(1 H, dd, J�11, 5 Hz, H-1b), 3.78 (1 H, m, H-4), 3.80 (1 H,m, H-2), 5.45 (1 H, dt, J�15, 6 Hz, H-6), 5.50 (1 H, dt,J�15, 6 Hz, H-7). 13C-NMR (125 MHz, CD3OD) d : 14.4 (C-19), 23.3 (C-18), 30.3—30.8 (C-9—C-16), 33.0 (C-17), 33.7 (C-8), 40.5 (C-3), 41.9 (C-5), 67.3 (C-1), 71.3 (C-4), 72.2(C-2), 127.2 (C-7), 134.4 (C-6).

RESULTS AND DISCUSSION

As part of our ongoing study on trypanocidal constituentsin plants, we have proceeded with the screening of trypanoci-dal activity of some Mexican plants. The list of crude extracts newly examined is shown in Table 1 with scientificnames, local names and parts examined. Annotated extractswere previously examined in ref. 2 against epimastigotesonly. The list is comprised of 41 families and 65 species. Inthe previous paper, we screened trypanocidal activity againstepimastigotes, stage form in the insect gut. This time we alsoexamined trypanocidal activity against trypomastigotes, in-fectious form in the mammalian blood stream. The activityagainst epimastigotes and trypomastigotes is shown by marks

���, ��, � and �. Each mark means MC100 value(mg/ml) to be �125, 125—250, 250—500 and�500, respec-tively. In the case of epimastigotes only one extract out of 71extracts showed strong activity and 10 extracts showed weakactivity. On the contrary, 39 extracts showed activity againsttrypomastigotes so far. It seems that trypomastigotes aremore sensitive than epimastigotes.

The MeOH extract of seeds of P. americana showed mod-erate activity against epimastigotes. When the MeOH extractwas separated into 3 fractions (frs. 1—3) by a polystyrenecolumn (Diaion HP-20), activity was observed in the lesspolar fraction 2 eluted with MeOH. Fraction 2 was subjectedto various kinds of chromatography to afford three 1,2,4-tri-hydroxyheptadec-16-ene derivatives (1—3), three 1,2,4-tri-hydroxyheptadec-16-yne derivatives (4—6), and two 1,2,4-trihydroxynonadecane derivatives (7, 8). Compounds 1—7were identified to be 1,2,4-trihydroxy-derivatives of C17 orC19 long-chain compounds by analyses of MS and NMRspectra as well as the comparison of physical data with thosereported from fruits and seeds of the same plants.7—12) Com-pound 8 was obtained as a solid. The molecular formula of 8,two hydrogen less than 7, C19H38O3, was confirmed by HR-FAB-MS. 1H- and 13C-NMR spectra of 8 showed the pres-ence of a 1,2,4-trihydroxy-moiety as 7 and a trans-disubsti-tuted double bond at d 5.45 (1 H, dt, J�15, 6 Hz) and 5.50(1 H, dt, J�15, 6 Hz) which corresponded to the carbon sig-nals at d 134.4 and 127.2, respectively. The former olefinicproton signal coupled with the methylene proton signals at d2.17 (2H, m), which were assigned as the signals of H-5 de-duced from correlations starting from H-1 signals in the1H–1H chemical shift correlation spectroscopy (COSY) spec-trum. Therefore, the olefinic linkage was located at C-6,7 and the structure of 8 was determined to be (E)-1,2,4-trihy-droxynonadec-6-ene.

MC100 values of these compounds and berberine chloride3)

are listed in Table 2. IC50 values against epimastigotes wereestimated as described in the preceding paper1) and areshown in Table 2.

In the case of withanolides1) and xanthones3,4) as well asmany MeOH extracts, trypanocidal activity against trypo-mastigotes is stronger than that against epimastigotes. How-ever, all 1,2,4-trihydroxyheptadecane and 1,2,4-trihydroxy-nonadecane derivatives obtained from seeds of P. americanashowed almost the same activity against epimastigotes andtrypomastigotes. The value of MC100(E)/MC100(T) rangedonly from 0.75 to 1.40. It might be due to the difference ofmode of action. Transformation of the terminal 16-ene groupto a 16-yne group reduced the activity. There was a small dif-ference of activity between 1-acetoxy/4-acetoxy and thelength of the carbon chain, C17/C19. Selective toxicity [IC50

(HeLa)/IC50 (T. cruzi)] of 1—8 is rather small (0.60—2.23)in the case of epimastigotes. Considering that these com-pounds are also contained in edible fruits, further assay in vivo is to be desired although the same small selective tox-icity is predicted in the case of infectious trypomastigotes.These compounds have been described previously as the bit-ter,7) antifungal,9,13) antibacterial,7,14) cytotoxic10) and insecti-cidal10) constituents in various parts of P. americana. This isthe first reference to the trypanocidal activity of these typesof compounds. Chemical investigation of the other activeplant extracts and bioassay in vivo are now in progress.

July 2005 1315

1316 Vol. 28, No. 7

Table 1. List of Plants Examined and Their Trypanocidal Activitya)

Family Scientific name Local name Partb) (T)

Acanthaceae Justicia spicigera Muicle G �Anacardiaceae Spondias mombin Ciruela amarilla S �Annonaceae Annona cherimola Chirimoya L ���c)

T ��c)

Annona muricata Guanabana L ��c)

T �Annona purpurea Chirimoya real L ��

T �Annona reticulata Anona L ���c)

T �Aristolochiaceae Aristolochia grandiflora Guaco R �

Aristolochia taliscana Guaco R �c)

Betulaceae Alnus acuminata Abedul LT �Bignoliaceae Parmentiera aculeata Cuajilote G �Boraginaceae Borago officinalis Borraja G �Campanulaceae Lobelia laxiflora Oxpaxihuatl G �Chenopodiaceae Chenopodium ambrosioides Epazote morado G �Compositae Bidens odorata Mosote blanco G �

Bidens pilosa Mozoquelite G �Heliopsis ongipes Chilcual R �Piqueria trinervia Altareina Rd) ���Stevia dictyophylla San marcos G �Tanacetum parthenium Santa Maria L �

Cucurbitaceae Maximowitzia sonorae Guareque R �Sechium edule Chayote G �

Elaeocarpaceae Muntingia calabura Capulín rojo L �Fr �

Equisetaceae Equisetum giganteum Cola de Caballo G �Ericaceae Gaultheria acuminata Axopaque LT ��Euphorbiaceae Croton draco Sangre de grado L �

Hura polyandra Haba o Habilla S �Phyllanthus nobilis Raiz de Cirueliya R ��

Gesneriaceae Kohleria deppeana Tlachichinola G �Guttiferae Calophyllum brasiliense Bari L ���c)

B �c)

Hyppocrateaceae Hippocratea excelsa Matapiojos R �Julianaceae Amphypterigium adstringens Cuachalalate B ��Labiatae Hyptis stellulata Te Milagro L ��

Marrubium vulgare Marrubio G ��Lauraceae Persea americana Aguacate S �c)

Leguminosae Acacia varnesiana Arloma LT �Brongniartia podalyrioides Hierba de la Víbora L �Lonchocarpus guatemalensis L �Lonchocarpus phenthaphylus Ld) ���e)

Lonchocarpus unifoliolatum L �Lonchocarpus oaxacensis R �Trigonella foenum-graecum Fenogreco S �

Liliaceae Smilax aristolochiifolia Zarzaparrilla R �Smilax xalapensis Alambrillo R �

Magnoliaceae Talauma mexicana Aguacote Fl �Marvaceae Malvaviscus arboreus Azocopacle L �Myrtaceae Psidium guajava Guayaba L ��Onagraceae Semelandra grandifolia Micle G �Papaveraceae Argemone platycerus Chicalote Fl �Piperaceae Piper auritum Acuyo L �Polypodiaceae Adiantum princeps Palmita G ��c)

Phlebodium aureum Lengua de ciervo R �Rosaceae Coluania mexicana Pez Chivo R �Rutaceae Casimiroa edulis Zapote blanco L �Simaroubaceae Castela tortuosa Chaparro Amargo R �Solanaceae Physalis philadelphica Tomate Fr �

Solanum hispidum Berenjena L ��Sterculiaceae Chiranthodendron pentadactylon Manita Fl �

Guazuma ulmifolia Guasima Fr �Taxodiaceae Taxodium macronatum Ahuehuete L ���Theaceae Ternstroemia sylvatica Tila Fl ��c)

Umbelliferae Eryngium carlinae Perejil G ��Foeniculum vulgare Hinojo L �

Urticaceae Urtica dioica Ortiga LT �Verbenaceae Lippia dulcis Hierba dulce L ��Zygophylaceae Larrea tridentate Gobernadora G �

a) MC100 values against trypomastigotes (T) in vitro. (�125 mg/ml, ���; 125—250 mg/ml, ��; 250—500 mg/ml, �; �500 mg/ml, �). Activity against epimastigotes is (�)unless otherwise mentioned. b) Extracted with MeOH unless otherwise mentioned. (S, seeds; L, leaves; T, twigs; R, roots; LT, leaves and twigs; G, ground parts; B, barks; Fl, flow-ers; Fr, fruits). c) MC100 value against epimastigotes is (�) in vitro. d) Extracted with CH2Cl2. e) MC100 value against epimastigotes is (���) in vitro.

Acknowledgements The authors are also grateful to Ms.Y. Sanches of Universidad Autonoma de Guadalajara for herassistance. We thank Ms. Y. Iwase and Mr. H. Hanazono for

NMR and MS measurements. This work was supported inpart by a Grant-in-Aid for Scientific Research (C) (No.14572028) from the Ministry of Education, Culture, Sports,Science and Technology, Japan.

REFERENCES AND NOTES

1) Part 4: Nagafuji S., Okabe H., Akahane H., Abe F., Biol. Pharm. Bull.,27, 193—197 (2004).

2) Abe F., Nagafuji S., Yamauchi T., Okabe H., Maki J., Higo H., Aka-hane H., Aguilar A., Jiménez-Estrada M., Reyes-Chilpa R., Biol.Pharm. Bull., 25, 1188—1191 (2002).

3) Abe F., Nagafuji S., Okabe H., Akahane H., Estrada-Muñiz E., Huerta-Reyes M., Reyes-Chilpa R., Biol. Pharm. Bull., 27, 141—143 (2004).

4) Abe F., Nagafuji S., Okabe H., Higo H., Akahane H., Biol. Pharm.Bull., 26, 1730—1733 (2003).

5) Errata in refs. 3 and 4. The host culture cells, LLC-MK2, should be re-vised from mouse kidney cells to rhesus monkey kidney cells.

6) Carmichael J., DeGraff W. G., Gazdar A. F., Minna J. D., Mitchell J.B., Cancer Res., 47, 936—942 (1987).

7) Brown B. I., J. Chromatogr., 86, 239—245 (1973).8) Kashman Y., Néeman I., Lifshitz A., Tetrahedron, 25, 4617—4631

(1969).9) Sugiyama T., Sato A., Yamashita K., Agric. Biol. Chem., 46, 481—485

(1982).10) Adikaram N. K. B., Ewing D. F., Karunaratne A. M., Wijeratne E. M.

K., Phytochemistry, 31, 93—96 (1992).11) Oberlies N. H., Rogers L. L., Martin J. M., McLaughlin J. L., J. Nat.

Prod., 61, 781—785 (1998).12) Brown B. I., J. Agr. Food Chem., 20, 753—757 (1972).13) Domergue F., Helms G. L., Prusky D., Browse J., Phytochemistry, 54,

183—189 (2000).14) Néeman I., Lifshitz A., Kashman Y., Appl. Microbio., 19, 470—473

(1970).

July 2005 1317

Table 2. Trypanocidal and Cytotoxic Activity of Compounds 1—8 andBerberine Chloride in Vitro (mM)

Compd. MC100(E)a) MC100(T)b) IC50(E)c) IC50(H)d)

1 183 244 82 492 213 228 107 1433 245 198 140 1924 276 337 114 2095 307 307 92 2056 327 294 123 2227 189 153 127 898 223 159 92 127

Berberine chloride 807 19 121

a) MC100 values against epimastigotes of T. cruzi. b) MC100 values against trypo-mastigotes of T. cruzi. c) IC50 values against epimastigotes of T. cruzi. d ) IC50 valuesagainst HeLa cells.

Chart 1