Abstract
In order to explore rationally the medical potential of the plant biodiversity of the Central and South American region as a source of novel antiparasitic molecules, a multinational Organization of American States (OAS) project, which included the participation of multidisciplinary research centers from Argentina, Bolivia, Colombia, Costa Rica, Guatemala, Nicaragua and Panama, was carried out during the period 2001-2004. This project aimed at screening organic plant extracts for antitrypanosomal, antileishmanial and antimalarial activities and subsequently isolating and characterizing bioactive molecules. Plants for antiparasitic screening were selected from a database of ethnomedical uses of Latin American plants (PlanMedia) based on the amount of biological and chemical information available in the literature. We report here the evaluation of 452 extracts from 311 plant species in vitro screens against Plasmodium falciparum, Leishmania mexicana, and Trypanosoma cruzi. Out of 311 species tested, 17 plants (5.4%) showed antiparasitic activities at IC50 values ≤ 10 µg/mL. The most active plants were Acnistus arborescens (L.) Schltdl. (Solanaceae) (leaf, EtOH, IC50: 4 µg/mL) Monochaetum myrtoideum Naudin (Melastomataceae) (leaf, MeOH, IC50: 5 µg/mL) and Bourreria huanita (Lex.) Hemsl. (Boraginaceae) (branch, EtOH, IC50: 6 µg/mL). These were selectively active against P. falciparum, L. mexicana and T. cruzi, respectively.
Introduction
Malaria, trypanosomiasis, and leishmaniasis have burdened the Latin American and Caribbean regions for centuries and have negatively influenced their ability to develop and become competitive societies in the current climate of globalization (CitationHotez et al., 2008). American trypanosomiasis (also known as Chagas disease) is caused by Trypanosoma cruzi and is the major endemic disease in Latin America. Malaria is a major global public health problem responsible for approximately one million cases per year in the Americas. The increasing global spread of drug resistance to most of the available and affordable antimalarial drugs is a major concern and requires innovative strategies for combating the disease. Leishmaniasis is a vector-borne disease, affecting 72 developing countries and in three Latin American countries. Visceral leishmaniasis caused by Leishmania donovani is the most severe form of Leishmania infections.
The current chemotherapy regimens for these parasitic diseases are limited and are not ideal because of the often associated severe side effects. The emergence of drug-resistant parasites presents an additional and major problem. All these facts underline the urgent need for the development of new, cheap, safe and easy-to-administer molecules for the treatment of these tropical diseases. Considering the great potential of Latin America in terms of its untapped plant biodiversity and rich traditional medicine, this study was undertaken to screen selected plants for in vitro antiplasmodial, antileishmanial, and antitrypanosomal activities and subsequently isolate the bioactive molecules.
Materials
Plant material
Plants were collected mainly from tropical forests throughout the seven countries (Argentina, Bolivia, Colombia, Costa Rica, Guatemala, Nicaragua and Panama). Their taxonomic identity was established by the botanists Martha Gattuso, M. Frecentese, Elisa Petenatti (Argentina); Rosy de Michel, Genevieve Bourdy, Andrés Roca (Bolivia); Ricardo Callejas, Edgar Linares, José Luis Fernández, Zaleth Cordero, Santiago Díaz, N.R. Salinas (Colombia), Luis Guillermo Acosta, Alexander Rodríguez (Costa Rica); Mario Veliz, Elfriede de Pöll, Juan José Castillo (Guatemala); Ricardo Rueda, Dania Paguaga, Hilario Mendoza, Nelson Toval y Miguel Garmendia (Nicaragua); Mireya Correa (Panamá). Voucher specimens are deposited in the corresponding National Herbarium in each country: Herbarium of the National University of San Luis (UNSL), Argentina; Herbarium of the Universidad Nacional de Rosario (UNR), Argentina; National Herbarium of Bolivia (HLP), Bolivia; National Colombian Herbarium (COL), Colombia; Herbarium of INBio(INB), Costa Rica; CEMAT-FARMAYA Ethnobotany Herbarium, (CFEH) Guatemala; Universidad Nacional Autónoma de Nicaragua-León (Herbario UNAN-León), (HULE), Nicaragua; and Herbarium of the Universidad de Panamá (PMA), Panama.
Selection of plants
A random list of plants from the ethnomedical database PlanMedia (CitationCIFLORPAN, 2004) of public information was submitted to a NAPRALERT (Natural Products Alert database, University of Illinois at Chicago) search for biological and chemical information. From that list, 311 plant species were prioritized according to the reported ethnomedical uses for three neglected diseases and lack of chemical and biological information.
Preparation of extracts
In general, plant material was macerated with 80% ethanol, methanol, dichloromethane, chloroform and hexane (24 h) for extraction (3 times). The plant extracts were filtered and concentrated in vacuo at < 40°C in a rotary evaporator and stored at -80°C until tested.
Biological assays
Antiplasmodial assay
Antiplasmodial activity was determined in a chloroquine-resistant P. falciparum strain (W2 Indochina) utilizing a novel microfluorimetric assay to measure the inhibition of the parasite growth based on the detection of the parasitic DNA by intercalation using Pico Green (CitationCorbett et al., 2004). The IC50 values were calculated from relative fluorescence units as compared with untreated controls. The parasites were maintained at 2% hematocrit in flat-bottom flasks (75 mL) in human red blood cells from O positive blood type donors with RPMI 1640 medium (Gibco BRL) supplemented with 10% O positive human serum. Chloroquine was used as a standard antimalarial drug and showed an IC50 value of 65-100 nM.
Antileishmanial assay
The study used a fluorimetric Leishmania mexicana (WHO-MOHM/B2/82/BELZ) axenic amastigote assay adapted from a novel antimalarial micro-method by CitationCorbett et al. (2004), to measure inhibition of the parasite growth based on the detection of parasitic DNA by intercalation with PicoGreen. Briefly, 25,000 amastigotes from axenic culture (CitationBates, 1993) were co-incubated with the test substances at 32°C in a CO2-free atmosphere for 72 h. The relative fluorescence units (RFU) were quantified with a fluorescence microplate reader (FLx800; Bio-Tek Instruments, Winooski, VT) at 485/20 nm excitation and 528/20 nm emission filters. Amphotericin B was used as a standard anti-leishmanial agent and had an IC50 value of 100 nM.
Antitrypanosomal assay
The recombinant Tulahuen clone C4 of Trypanosoma cruzi, which expresses β-galactosidase (βGal) as a reporter enzyme was used in the assay (CitationBuckner et al., 1996). The method was based on the growth inhibition effect of the test samples on trypomastigote, the bloodstream form of the parasite. The resulting color from the cleavage of chlorophenol red-β-galactopyranoside (CPRG) by βGal, expressed by the parasite, was measured at 570 nm as indirect measurement of parasite growth. The inhibitory concentration of 50% growth (IC50) as compared with the untreated control was calculated from the optical density values. Assays were conducted at 37°C under an atmosphere of 5% CO2/95% air mixture. Nifurtimox was tested as a standard antitrypanosomal agent and had an IC50 value in the range of 10 nM.
Results
Antiparasitic activities of extracts
Four hundred fifty-two extracts were prepared from 311 plants and were first tested at the concentration of 50 µg/mL on a chloroquine-resistant P. falciparum strain (W2 Indochina), Leishmania mexicana (WHO-MOHM/B2/82/BELZ), and Trypanosoma cruzi. Of plants tested at this stage 49% belong to the major families such as Asteraceae, Piperaceae, Rubiaceae, Solanaceae, and Fabaceae. Ninety-four extracts representing 86 plants, 68 genera and 39 families showed IC50 ≤ 50 µg/mL (). The in vitro antitrypanosomal, antiplasmodial and antileishmanial activities of the extracts were qualified as most active when IC50 values were ≤ 10 µg/mL.
Table 1. Active antiparasitic plants.
Antitrypanosomal activity
Nine plant extracts showed IC50 ≤ 10 µg/mL. The most active plants against T. cruzi were Acnistus arborescens (leaf, EtOH), Scoparia dulcis L. (Scrophulariaceae) (whole plant, EtOH) and Maianthemum paludicola La Frankie (Convallariaceae) (whole plant, MeOH), with IC50 values of 4, 4 and 5 µg/ml, respectively. Chromolaena leivensis (Hieron.) R.M. King & H. Rob (Asteraceae) (aerial parts, EtOH) showed antitrypanosomal activity at IC50 value of 8 µg/mL, whereas Annona muricata L. (Annonaceae) (leaf, EtOH), Argemone subfusiformis G.B. Ownbey (Papaveraceae) (fruit, MeOH) and Caesalpinia paraguariensis (D. Parodi) Burkart (Fabaceae) (leaf, EtOH), Piper barbatum Kunth (Piperaceae) (leaf, EtOH) and Piper holtonii C. DC. (Piperaceae) (root, EtOH) displayed the same activity at IC50 value of 10 µg/mL. The antitrypanosomal activity of these species has not been reported previously in the literature except in the case of Annona muricata, which has been used in the folk medicine of Nicaragua for diarrhea (CitationCoee & Anderson, 1996) and for fever in Colombia (CitationBlair et al., 1991). Its antitrypanosomal activity against epimastigotes was reported previously (CitationAbe et al., 2005). The 95% ethanol extract of seeds of Annona cherimola Mill. (Annonaceae) displayed antitrypanosomal activity against T. cruzi (CitationKim et al., 2007). Stem bark (petroleum ether extract) of Annona senegalensis Pers. (Annonaceae) showed activity against Trypanosoma brucei at MIC of 19 µg/mL (CitationFreiburghaus et al., 1996).
The Izoceño-guarani use Argemone subfusiformis against cough, cold, and flu in Bolivia (CitationBourdy, 2002), and intestinal worms in Argentina (CitationMartínez Crovetto, 1981).
Chromolaena leivensis has been used traditionally as febrifuge (CitationMoreno & Tinjaca, 1986). A related species C. christieana (Baker) R.M. King & H. Rob. (Asteraceae) (stem and bark) showed the highest percentage of lysis on bloodstream forms of T. cruzi at a concentration of 250 μg/mL (CitationRojas de Arias et al., 1995).
Piper holtonii and P. barbatum have been used in Colombian traditional medicine for fever and malaria (CitationGarcía-Barriga, 1975). Five chromenes were isolated from Piper gaudichaudianum Kunth and Piper aduncum L. (Piperaceae) and displayed in vitro activity against epimastigote forms of T. cruzi (CitationBatista et al., 2008). Neolignans isolated from leaves of Piper regnellii (Miq.) C. DC. var. pallescens (C. DC.) Yunck (Piperaceae) showed in vitro antiproliferative effects against T. cruzi (CitationLuize et al., 2005, Citation2006). Although Piper glabratum Kunth and P. acutifolium Ruiz & Pav. (Piperaceae) were not active in our screening, two benzoic acid derivatives with moderate activity against P. falciparum were isolated and reported by CitationFlores et al. (2008).
Scoparia dulcis (leaves), called “tupeicha” in Bolivia (Licorice weed or Sweet broom), has been used in traditional medicine of the Guarani for dysentery, diarrhea, stomachache and fever (CitationBourdy, 2002).
Caesalpinia paraguariensis, commonly known as “ivirayepiro”, has been used traditionally by the Guarani ethnic group in Paraguay to treat dysentery, bloody diarrhea, furuncles, fever, stomach ache and body ache (CitationBourdy, 2002). Norcaesalpinin E, a furanocassane-type diterpene, isolated from Caesalpinia crista L. (Fabaceae) (seed kernels) showed a higher antimalarial activity (IC50 value = 0.090 µM) than chloroquine (CitationLinn et al., 2005; CitationKalauni et al., 2006). Five out of nine active antitrypanosomal plants from this study were previously reported for their ethnomedical uses to treat symptoms related to Chagas disease.
Antiplasmodial activity
Five plant extracts showed activity at IC50 ≤ 10 µg/ mL. The most active plants were Monochaetum myrtoideum (leaf, EtOH), Bourreria spathulata (Miers) Hemsl. (Boraginaceae), (leaf, EtOH), Polygonum acuminatum Kunth (Polygonaceae) (leaf, MeOH), Clematis campestris A. St.-Hil. (Ranunculaceae) (flower, MeOH) and Terminalia triflora (Griseb.) Lillo (Combretaceae) (aerial parts, MeOH) at IC50 values of 5, 8, 8, 9 and 9 µg/ml, respectively. There is no report on the chemical composition of Terminalia triflora, but other species such as Terminalia glaucescens Planch. ex Benth. (Combretaceae) used in West African traditional medicine for malaria has shown in vitro antiplasmodial activity (IC50 2.34-4.83 µg/ml) against various Plasmodium falciparum strains (CitationMustofa et al. 2000). An aqueous extract of Terminalia macroptera Guill. & Perr. (Combretaceae) (used in traditional medicine in Burkina Faso) displayed activity against Plasmodium falciparum chloroquine-resistant W2 strain (IC50 = 1 µg/ml) (CitationSanon et al., 2003). Polar extracts of T. glaucescens (leaf and stem) showed antiplasmodial activity against chloroquine-resistant P. falciparum at IC50 values between 0.4-8 µg/ml (CitationMustofa et al., 2000).
Species of Polygonum, Polygonum senegalense Meisn. (Polygonaceae), displayed antiplasmodial activity due to the presence of 9-hydroxyhomoisoflavanone in aerial exudates (CitationMidiwo et al., 2007). Polar extracts of Polygonum multiflorum Thunb. (Polygonaceae) root showed antiplasmodial activity against chloroquine-resistant P. falciparum (CitationTran et al., 2003). Species of Monochaetum from Colombia have been used in traditional medicine for fever and paludism (CitationGarcía-Barriga, 1975).
This is the first report of the antimalarial activity of Monochaetum myrtoideum, Bourreria spathulata, Polygonum acuminatum, and Terminalia triflora. The bioactive compounds responsible for this activity have not yet been reported. The ethnomedical uses of only one out of five antiplasmodial plants correlate with their observed biological activity.
Antileishmanial activity
Three plant extracts showed activity at IC50 value of ≤ 10 µg/mL. The most active plants were Bourreria huanita (bark, EtOH), Mikania periplocifolia Hook. & Arn. (Asteraceae) (aerial parts, MeOH) and Parietaria debilis G. Forst. (Urticaceae) (aerial parts, MeOH) at IC50 values of 6, 7 and 8 µg/ml, respectively. This is the first report of the antileishmanial activity of these three plants; no compound has been identified as responsible for this biological activity so far.
Mikania periplocifolia is used as febrifuge in Argentina (CitationZardini, 1984). A related species, M. congesta DC. (Asteraceae), has also been reported with the same ethnobotanical use in Peru (CitationRoumy et al., 2007). Another species, Mikania, M. glomerata Spreng. (Asteraceae) showed significant effects against axenic amastigote and promastigote forms of Leishmania (L.) amazonensis at a concentration of 100 μg/mL with a percentage of growth inhibition between 49.5 and 99% (CitationLuize et al., 2005). Parietaria debilis is used traditionally for washing blisters in Argentina (CitationMartínez Crovetto, 1981). The ethnomedical uses of two out of three antileishmanial plants are in agreement with the results of this study.
Conclusions
This study has identified a number of plant extracts () that have shown in vitro antiparasitic activities, based on the selection of plants listed in the database PLANMEDIA. The results of the antiparasitic screening revealed that 2 out of 17 active plants belong to the genus Piper. In the species examined, their ethnomedical uses show a good correlation with the observed antiparasitic activity. These are under study to isolate active compounds.
Acknowledgements
The authors are thankful to J. Gonzalez, L. Ureña, L. Abrego, Z. Capitan Barrios for antiparasitic testing. Thanks are also due to the National Environmental Authority of Panama for granting permission to collect plants in national parks. We also thank all taxonomists involved in the project who identified plants.
Declaration of interest
This work was supported by the Organization of American States (OAS) through the multinational project “Aprovechamiento de la Flora Regional como Fuente de Moléculas Antifúngicas, Antiparasitarias y Anticancer” (SEDI/AICD/106/01).
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