Abstract
Context: Malaria is still a major public health problem. The biodiversity of the tropics is extremely rich and represents an invaluable source of novel bioactive molecules. For screening of this diversity more sensitive and economical in vitro methods are needed, Flora of Panama has been studied based on ethnomedical uses for discovering antimalarial compounds.
Objective: This review aims to provide an overview of in vitro screening methodologies for antimalarial drug discovery and to present results of this effort in Panama during the last quarter century.
Methods: A literature search in SciFinder and PubMed and original publications of Panamanian scientists was performed to gather all the information on antimalarial drug discovery from the Panamanian flora and in vitro screening methods.
Results and conclusions: A variety of colorimetric, staining, fluorometric, and mass spectrometry and radioactivity-based methods have been provided. The advantages and limitations of these methods are also discussed. Plants used in ethnomedicine for symptoms of malaria by three native Panamanian groups of Amerindians, Kuna, Ngöbe Buglé and Teribes are provided. Seven most active plants with IC50 values < 10 μg/mL were identified Talisia nervosa Radlk. (Sapindaceae), Topobea parasitica Aubl.(Melastomataceae), Monochaetum myrtoideum Naudin (Melastomataceae), Bourreria spathulata (Miers) Hemsl.(Boraginaceae), Polygonum acuminatum Kunth (Polygonaceae), Clematis campestris A. St.-Hil. (Ranunculaceae) and Terminalia triflora (Griseb.) Lillo (Combretaceae). Thirty bioactive compounds belonging to a variety of chemical classes such as spermine and isoquinoline alkaloids, glycosylflavones, phenylethanoid glycosides, ecdysteroids, quercetin arabinofuranosides, clerodane-type diterpenoids, sipandinolid, galloylquercetin derivatives, gallates, oleamide and mangiferin derivatives.
Introduction
The emergence and spread of strains of Plasmodium falciparum resistant to available antimalarial drugs require a constant monitoring of parasite susceptibility to antimalarial drugs and a concerted effort towards the search for new potent antimalarials. Over the years, ethnomedicine has been a potential lead for the discovery of antimalarial compounds and templates for the synthesis of novel antimalarial molecules. The first step in the antimalarial drug discovery process is to evaluate the antimalarial activity of plant extracts or the test compounds in vitro systems using well-characterized strains of P. falciparum. This review provides a comprehensive study of all available in vitro screening methodology and a summary of antimalarial activity of Panamanian flora during the last quarter century. Previous reviews on in vitro screening methods have been published by CitationFidock et al. (2004) and CitationKalra et al. (2006).
In vitro screens of potential antimalarials
Radioactivity-based assay
The 3H-hypoxanthine incorporation is the method most commonly used to determine in vitro growth inhibition of P. falciparum in human erythrocytes (CitationDesjardins et al. 1979). [3H]-hypoxanthine is used (which is taken up by parasite for purine salvage pathway and DNA synthesis) to determine the level of P. falciparum growth inhibition. The disadvantages of this method are that it is costly, quite complex and requires radioactive material which poses safety and disposal problems especially in developing countries and, therefore, is problematic for resource-poor institutions or for high-throughput screening (HTS).
Light microscopy-based assay
Another method is the Giemsa-stained slide method which is based on the incubation of test compounds with P. falciparum followed by a comparison of parasitaemias of treated and control parasites by counting Giemsa-stained parasites by light microscopy. Even though it is low-cost, it can be used only to test small number of compounds (CitationDesjardins, 1984).
Colorimetric assays
Another established assay involves the colorimetric detection of lactate dehydrogenase. This assay is based on the observation that the lactate dehydrogenase of P. falciparum has the ability to rapidly use 3-acetyl pyridine NAD (APAD) as a coenzyme in the reaction leading to the formation of pyruvate from lactate. The main drawback of this method is that it is less standardized (CitationMakler & Hinrichs, 1993) and involves multiple steps which make it unsuitable for high-throughput antimalarial drug screening.
Another colorimetric method based on P. falciparum thioredoxin reductase activity (TrxR) has been used to screen antimalarial compounds of natural and synthetic origins. In this assay, 5,5′-dithiobis(2-nitrobenzoate) (DTNB) is used as the substrate in TrxR assay buffer (100 mM potassium phosphate, 2 mM EDTA, pH 7.4), and the production of TNB− anions (ϵ412 nm = 13.6 mM−1 cm−1) is monitored spectrophotometrically (CitationSturm et al., 2009).This assay has the advantage that TrxR activity can also be determined using its physiological substrate, thioredoxin (Trx).
A mechanistic type of assay is the determination of inhibition of β-hemozoin formation in the food vacuole of the malaria parasite. The 60-min assay is based on UV absorption of β-hemozoin at 405 nm that allows the quantitation of hematin concentration in hematin/β-hematin mixtures in the presence of test compounds. This simple assay was adapted for high throughput colorimetric screening, allowing visual identification of β-hematin inhibitors (CitationNcokazi& Egan, 2005).
A protocol for in vitro hemozoin formation assay to be used for in vitro screening of synthetic and natural compound libraries and for the identification of novel inhibitors of β-hematin formation was developed by CitationTripathi et al. (2005). This colorimetric assay comprises minimal number of steps, particularly for complete removal of aggregated monomeric heme and accurate quantification of nascent β-hematin. The use of commercially available oleoyl glycerol as a catalytic agent ensures reproducibility and high sensitivity of the assay. The assay is robust, inexpensive, and provides sensitivity and applicability to automated and configured to a high-throughput format.
Flow cytometry-based assays
Flow cytometry takes advantage of the fact that human erythrocytes lack DNA. In this technology, parasites are fixed after appropriate period of incubation with test compounds, and then either the parasitized cells are stained with hydroethidine (which is metabolized to ethidium) or the parasite nuclei are stained with 4′, 6-diamidino-2-phenylindole (DAPI). Counts of treated and control cultures are then obtained by flow cytometry. Appropriate gating can also allow one to distinguish different parasite stages in erythrocyte. This relatively simple assay is amenable to high throughput, but requires expensive equipment (Citationvan der Heyde et al., 1995). Flow cytometry-based assays using green fluorescent protein (GFP) containing D10 P. falciparum parasites proved sensitive and highly reproducible for quantifying the growth-inhibitory activity of antimalarial compounds, with superior reproducibility to light microscopy, and are suitable for high-throughput applications (CitationWilson et al., 2010).
Fluorimetric assays
Non-radioactive DNA stains have been reported for measurement of parasite growth in a short-term assay using a 96-well format, among them a microfluorimetric assay to measure the inhibition of P. falciparum was based on the detection of parasitic DNA by intercalation with PicoGreen® (CitationSinger et al., 1997).The method was used to determine parasite inhibition profiles and 50% inhibitory concentration values of potential antiplasmodial compounds (CitationCorbett et al., 2004). Values for parasite inhibition with known antimalarial drugs using the PicoGreen® assay were reported to be comparable with those determined by the standard method based upon the uptake of [3H]-hypoxanthine and the Giemsa stain microscopic technique. The PicoGreen® assay is rapid, sensitive, reproducible, easily interpreted, and ideally suited for screening of large numbers of samples for antimalarial drug development.
The assay monitors DNA content by the addition of the fluorescent dye DAPI as a reporter of blood-stage parasite growth (CitationBaniecki et al, 2007). This DAPI P. falciparum growth assay was used to measure the 50% inhibitory concentrations (IC50s) of a diverse set of known antimalarials. The resultant IC50 values compared favorably with those obtained in the [3H]-hypoxanthine incorporation assay. This P. falciparum growth assay is technically simple, robust, and compatible with the automation necessary for HTS.
The SYBR Green 1 assay according to method of CitationSmilkstein et al. (2004) is based on the use of SYBR® Green I nucleic acid gel stain. Parasite growth is determined by using SYBR Green I, a dye with marked fluorescence enhancement upon contact with Plasmodium DNA. Although, PicoGreen®, like ethidium bromide, is an intercalating dye (CitationSinger et al., 1997) while SYBR Green I appears to be significantly less mutagenic than ethidium bromide (CitationSYBR Safe® Case Study 2006). SYBR Green I appears to be the least expensive assay. The microfluorimetric assay using SYBR Green I is a cost-effective, safe alternative for antimalarial drug screening and a viable technique that may facilitate antimalarial drug discovery process especially in developing countries where available funding is limited.
Mass spectrometry-based binding method
An ultrafiltration and liquid chromatography and mass spectrometry (UF and LC/MS) based binding assays were developed for P. falciparum thioredoxin (PfTrxR) and glutathione (PfGR) reductases to identify potential lead compounds for malaria drug discovery. In the MS-based binding assays, the relative binding affinity of compounds for both the enzymes was tested under competitive and non-competitive incubation conditions. The current method developed has a potential for automated HTS screening to rapidly determine the binding affinity of ligands for these enzymes for pure compounds and complex mixtures of natural products.
Assays to measure drug resistance
To assess the effects of combining compounds, isobologram analysis (CitationBerenbaum, 1978; CitationOhrt et al., 2002) can be performed to assess whether two compounds are additive, synergistic or antagonistic. This is conducted using standard dose–response assays over a range of individual drug concentrations, using either a checkerboard technique (CitationCanfield et al., 1995) or fixed-ratio methods (CitationOhrt et al., 2002).
The most commonly used method for the antimalarial in vitro testing for resistance is Micro-test (Mark III) (CitationWHO, 2004). It provides information on the quantitative drug response of P. falciparum irrespective of the patient’s immune system. The in vitro test can be carried out with several drugs, in a Micro test kit with 12 × 8 wells, predosed with chloroquine, mefloquine, quinine, amodiaquine, artemisinin, sulfadoxine (SDX)/pyrimethamine (PYR) and pyrimethamine (PYR). Patient’s blood sample is inoculated in the wells and incubated with suitable medium. The number of schizonts with three or more nuclei out of a total of 200 asexual parasites is counted and compared with control wells.
For monitoring the level and spread of resistance, molecular diagnostic methods for detecting resistant parasite have been proposed (CitationPlowe et al., 1995). These methods are suitable for using a large number of samples in malaria endemic areas and have major advantage over in vitro tests that require parasite cultivation which takes days to perform (CitationPlowe et al., 1996). These molecular tools are based on the detection by polymerase chain reaction of point mutation in the parasite genome responsible for in vitro resistance. Among the different classes of antimalarial screening assays the following advantages and disadvantages are common.
The in vitro methods are rapid, precise and efficient. They allow handling of a large number of compounds and permit better assessment of intrinsic activity of a drug. Moreover, synergism and antagonism with drug combinations can also be studied. They do have certain limitations which include: less reproducibility of pharmacokinetic effects, selection of toxic compounds, lack of clinical correlation, need for infrastructure and expertise and difficulty in studying drugs that act through active metabolites.
Antimalarial compounds from Panamanian flora
Previous reviews on potential of medicinal plants as sources of new antimalarial compounds have been written by CitationKvist et al. (2006), CitationTaylor et al. (2006), CitationKaur et al. (2009), CitationMuthaura et al. (2011), CitationWillcox, 2011, CitationBero et al. (2011), Martin CitationPohlit et al. (2011).
Flora of Panama is one of the richest in the world with an estimate of 9965 different species of vascular plants and 2113 genera (CitationCorrea et al., 2004; Correa, 2011). Out of total number of species, 1327 (13.3%) are endemic. Seven families of the Panamanian flora have more than 50 genera () and the genera Epidendrum, Piper, Anthurium, Elaphoglossum, Miconia, Psychotria, Philodendron, Peperomia have more than 100 species () (CitationCorrea et al., 2004; Correa, 2011). During the last quarter century, a total of approximately 390 compounds with various biological activities from 86 plants have been isolated, of which 160 are new to the literature. The list of medicinal plants used for symptoms of malaria by Kuna, Ngöbe Buglé and Teribe Indians of Panama based on work CitationGupta et al. (1993; Citation2005), CitationJoly et al. (1987; Citation1990), is depicted in .
Table 1. Plant families with higher number of genera in Panamanian flora, 50 or more.
Table 2. Genera with higher number of species in Panamanian flora, 100 or more.
Table 3. Medicinal plants used for symptoms of malaria by Kuna, Ngobe Buglé, and Teribe Indians of Panama.
A literature search in SciFinder and PubMed and original publications of Panamanian scientists was performed to gather all the information on antimalarial drug discovery from the Panamanian flora.
The Training in Tropical Diseases/World Health Organization (TDR/WHO) project was carried out from 2003 to 2005 in a 0.1-ha biodiversity plot in the Altos de Campana National Park to discover novel active antiparasitic and larvicidal compounds in Panamanian plants. One hundred and fifty organic plant extracts representing 43 families, 73 genera, and 93 species were subjected to antimalarial screening against P. falciparum. W2, chloroquine-resistant strains. Of these 150 plant extracts, two (1.3%) (Talisia nervosa Radlk. (Sapindaceae) and Topobea parasitica Aubl.(Melastomataceae) showed significant antimalarial activity (IC50 values < 10 µg/mL). Ethyl gallate (1) and methyl gallate (2) were isolated from stems of T. nervosa by bioassay-guided fractionation. Both 1 and 2 showed weak in vitro antiplasmodial activity against P. falciparum. (IC50 35.3 µM and IC50 38.0 µM, respectively), but they were less active than chloroquine (IC50 0.088 µM) (CitationCalderón et al., 2006).
Within the framework of 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, research was carried out during the period 2001–2004. Four hundred and 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). Of plants tested at this stage 49% belong to the major families such as Asteraceae, Piperaceae, Rubiaceae, Solanaceae, and Fabaceae. Five plant extracts showed activity at IC50 ≤ 10 μg/mL. The most active plants were Monochaetum myrtoideum Naudin (Melastomataceae) 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 5.0, 8.0, 8.0, 9.0 and 9.0 μg/mL respectively (CitationCalderón et al., 2010).
Four active quassinoids, ailanthone (3), 2′-acetylglaucarubinone (4), holacanthone (5) and glaucarubinone (6), were identified from dried fruits of Simarouba amara Aubl. (Simaroubaceae) used in ethnomedicine for malaria. They showed in vitro antiplasmodial activity against P. falciparum K1 with IC50 values of 9.0, 8.0, 7.0, 4.0 ng/mL, respectively. These compounds were tested in vivo against P. berghei and displayed ED50 values between 0.86 and 2.19 mg/kg/day. Holacanthone showed lesser toxicity (1 death out of 18 mice) than the other four quassinoids (CitationO’Neill et al., 1988).
A new benzoquinone (1-hydroxybenzoisochromanquinone) (7) and benz[g]isoquinoline-5,10-dione (8) were isolated from methanol extract of stems and roots of Notopleura camponutans (Dwyer & M.V. Hayden) C.M. Taylor (Rubiaceae) which displayed antimalarial activity against P. falciparum (chloroquine-resistant strain K-1) with IC50 values of 2.66 and 0.84 μg/mL, respectively (CitationSolís et al, 1995).
From a lipophilic extract of the leaves of Siparuna andina (Tul.) A. DC. (Monimiaceae), which exhibited antiplasmodial activity in vitro, two new compounds were isolated: sipandinolide, a compound with a novel type of carbon skeleton and (−)-cis-3-acetoxy-4′,5,7-trihydroxyflavanone. (−)-cis-3-acetoxy-4′,5,7-trihydroxyflavanone (9) which showed moderate antiplasmodial activity against P. falciparum poW with the IC50 value of 24.3 mg/mL whereas sipandinolide was inactive (CitationJenett-Siems et al., 2000).
Bioassay-guided fractionation of the leaves from Andira inermis (W. Wright) Kunth ex DC. (Fabaceae) was undertaken as part of a screening program carried out by CitationKraft et al. (2001) to verify the traditional use of herbal remedies against malaria. Among the isolated phenolic compounds, two novel 2-arylbenzofuran-3-carbaldehydes, andinermal A (10) exhibited the strongest antiplasmodial activity with IC50 values of 2.3 μg/mL against chloroquine-sensitive strain poW and 3.9 μg/mL against chloroquine-resistant strain Dd2 and andirnermal C (11) was slightly less active (5.9 μg/mL [poW], 6.3 μg/mL [Dd2]). Two isoflavones calycosin(3′, 7-dihydroxy-4′-methoxyisoflavone) (12) and genistein (4′,5,7-trihydroxyisoflavone) (13) isolated from stems of A. inermis have been shown to possess in vitro activity against the chloroquine-sensitive strain poW (IC50 4.2 μg/mL, 2.0 μg/mL) and the chloroquine-resistant (IC50 9.8 μg/mL, 4.1 μg/mL) clone Dd2 of P. falciparum (CitationKraft et al., 2000).
Methanol extracts of stem bark and leaves of Albizia adinocephala (Donn. Sm.) Britton & Rose ex Record (Fabaceae) inhibited the malarial enzyme plasmepsin II. The two new spermine alkaloids budmunchiamines L4 (14) and L5 (15) were isolated from stem bark of A. adinocephala which displayed mild activity against plasmepsin II with IC50 values of 14 and 15 μM, respectively (CitationOvenden et al., 2002).
Leaves of Cornutia pyramidata L. (Lamiaceae) are traditionally used against fever in Panama. CitationJenett-Siems et al. (2003) isolated the neo-clerodane-type diterpenoids cornutins C–F and evaluated their in vitro antiplasmodial activities. Of these, only cornutins C (16) and D (17) possessed slight activity against two different strains of P. falciparum (poW, Dd2), with the acetylated cornutin D being the least active one [IC50 23.1 μg/mL (56.6 mM, poW); 39.5 μg/mL (96.8 mM, Dd2)], whereas cornutin C showed IC50 values of 14.6 (36.9 mM, poW) and 21.5 μg/mL (58.7 mM, Dd2).
A decoction of the leaves of Siparuna pauciflora (Beurl.) A. DC. (Monimiaceae) is used by certain Indians in Panama against fever. The evaluation of the antiplasmodial activity of the isolated compounds against two strains of P. falciparum (poW, Dd2) showed a moderate activity of norboldine (18) [IC50 values: 3.1 μg/mL (pow), 5.4 μg/mL (Dd2)], whereas the other aporphines as well as the sesquiterpenoids proved to be inactive (CitationJenett-Siems et al., 2003).
Bioguided fractionation of the roots of Socratea exorrhiza (Mart.) H. Wendl. (Arecaceae) was carried out by CitationValdés (2005). S. exorrhiza was collected by the botanist Alex Espinosa in Parque Nacional Altos de Campana, sendero Panamá (N 08° 40′W 079 55′) on June 2, 2004. The taxonomic identification was confirmed by Mireya Correa, Director of the Herbarium of the University of Panama, and the voucher specimen (Florpan N° 6541) were deposited in the Herbarium of the University of Panamá (PMA). The ethanol extract of S. exorrhiza roots was patritioned according to Kupchan method (CitationHussein et al., 2003). The ethanol extract of S. exorrhiza (roots) displayed antiplasmodial activity against P. falciparum W2 (IC50: 14 μg/mL). Ethyl acetate and methanol fractions showed antiplasmodial activity with IC50 values of 12 and 24 μg/mL, respectively. Two compounds (3-O-β-sitosterolglucoside and 5,7,3′-trihydroxy-6-4′-dimethoxyflavanone) were isolated from the methanol fraction but showed no antiplasmodial activity in the PicoGreen® assay.
The known lignan, 7′-epi-sesartemin(19) was isolated from the leaves of Piper fimbriulatum C. DC (Piperaceae) along with two known lignans and a known flavone. Compound 19 displayed weak antiplasmodial activity (IC50 value of 7.0 μg/mL) (CitationSolís et al., 2005).
One new prenylated xanthone, 1,5-dihydroxy-3-methoxy-4-isoprenylxanthone (20), along with four previously known prenylated xanthones, ananixanthone, 1,3,7-trihydroxy-2,4-diisoprenylaxanthone (21), 8-desoxygartanin, and toxyloxanthone A (22), have been isolated from the MeOH liquid–liquid partition fraction of the crude extract of methanol–ethyl acetate extract of fresh and mature leaves of Chrysochlamys tenuis Hammel (Clusiaceae). Compound 1,5-dihydroxy-3-methoxy-4-isoprenylxanthone (20) showed moderate activity (31 ± 9 mM) against a chloroquine-resistant strain of P. falciparum, and compounds 1,3,7-trihydroxy-2,4-diisoprenylxanthone (21) and toxyloxanthone A (22) showed the highest antimalarial potency, IC50: 20 ± 2 and 16 ± 4 mM, respectively (CitationMolinar-Toribio et al., 2006).
The flavonol arabinoside, 5-galloylquercetin-3-O-R-l-arabinofuranoside (23), was isolated from the young leaves of Calycolpus warszewiczianus O. Berg. (Myrtaceae). The compounds were tested in vitro against a chloroquine-resistant strain of P. falciparum, Leishmania mexicana, and Trypanosoma cruzi parasites. Compound 23 demonstrated weak activity against a chloroquine-resistant strain of P. falciparum (14.5 μM) and no cytotoxicity was detected against mammalian cells below 100 μg/mL (CitationTorres-Mendoza et al., 2006).
The boiled juice of the whole plant Stachytarpheta cayennensis (Rich.) Vahl (Verbenaceae), is used in folk medicine in different countries of Latin-America to treat symptoms of malaria. From the antimalarial ethyl acetate extract, five phenylethanoid glycosides could be identified (CitationFroelichet al., 2008). Three of these phenylethanoid glycosides, leucosceptoside A, martynoside and jionoside D were detected for the first time in the genus Stachytarpheta. The two remaining isolated compounds, namely acteoside and iso-acteoside, were already reported in S. cayennensis (CitationAdebajo et al., 2007).
A new O-galloyl-C-glycosylflavone, 2″, 6″-O-digalloylvitexin (24), along with a known glycosylflavones (25) have been isolated from methanol extract of Clidemia sericea D. Don (Melastomataceae) leaves, and one known glycosylflavone (26) have been isolated from leaves of Mosquitoxylon jamaicense Krug & Urb. (Anacardiaceae). Compounds 24, 25, and 26 showed mild antimalarial activity (24 ± 1, 38 ± 2, and 44 ± 1 mM, respectively) against a chloroquine-resistant P. falciparum strain (CitationMontenegro et al., 2007).
Bioactivity-guided fractionation of the methanol extract from the leaves of Arrabidaea patellifera (Schltdl.) Sand with (Bignoniaceae) afforded mangiferin (27), isomangiferin, and six new derivatives (3′-O-p-hydroxybenzoylmangiferin (28), 3′-O-trans-coumaroylmangiferin (29), 6′-O-trans-coumaroylmangiferin,3′-O-trans-cinnamoylmangiferin,3′-O-trans-caffeoylmangiferin, and 3′-O-benzoylmangiferin). Compounds 27, 28 and 29 were relatively active in vitro against P. falciparum with IC50 values of 23.8, 26.5, 18.1 μM, respectively (CitationMartin et al., 2008).
The ethanol extract of Dichorisandra hexandra (Aubl.) Standl. (Commelinaceae) presented in vitro activity against Plasmodium falciparum W2 (IC50< 1 mg/mL). Two known inactive ecdysteroids (20-hydroxyecdysone 1 and muristerone A) were isolated from D. hexandra (Chung et al., 2009).
In the study carried out by CitationMunigunti et al. (2011), detannified methanol extracts from Guatteria recurvisepala R.E. Fr. (Annonaceae) (leaves), Topobea watsonii Cogn. (Melastomataceae) (stems and branches) and Licania kallunkiae Prance (Chrysobalanaceae) (stems) showed >50% inhibition of the growth of chloroquine-resistant P. falciparum strain (W2 Indochina) at 100 µg/mL. These extracts were screened for ligands to PfTrxR using ultrafiltration and liquid chromatography−mass spectrometry-based binding experiments. The PfTrxR ligand identified in the extract of G. recurvisepala was oleamide (30), which exhibited a relative binding affinity of 3.5-fold when incubated with 1 µM PfTrxR and the corresponding in vitro activity against P. falciparum strainK1 (IC50 4.29 μg/mL). shows all the structures of the compounds reported here.
Figure 1. Antimalarial compounds isolated from Panamanian plants.
Conclusions
This review provides in a summarized form an overview of various in vitro screening methodologies for antimalarial drug discovery. A variety of colorimetric, staining, fluorometric, and newer mass spectrometry and radioactivity-based methods have been provided. The advantages and limitation of these methods are also discussed. Plants used in ehtnomedicine for symptoms of malaria by three native groups of AmeriIndians, Kuna, Ngöbe Buglé and Teribes, based on ethnomedical published surveys, are provided. Screening of 652 organic plant extracts using these in vitro methods has permitted us in the identification of seven most active plants with IC50 values < 10 μg/mL: T. nervosa Radlk. (Sapindaceae) and T. parasitica Aubl. (Melastomataceae), M. myrtoideum Naudin (Melastomataceae), B. spathulata (Miers) Hemsl. (Boraginaceae), P. acuminatum Kunth (Polygonaceae), C. campestris A. St.-Hil. (Ranunculaceae) and T. triflora (Griseb.) Lillo (Combretaceae). Plants were also selected based on folk use for bioguided fractionation. Thirty bioactive compounds belonging to a variety of chemical classes such as spermine alkaloids, glycosylflavones, phenylethanoid glycosides, ecdysteroids, gallates, quercetin arabinofuranosides, isoquinoline alkaloids, clerodane-type diterpenoids, sipandinolid with novel carbon skeleton, galloylquercetin derivatives, gallates, oleamide and mangiferin derivatives. It is clearly evident that the flora of Panama is extremely rich and much remains to be done.
Acknowledgements
Thanks are due to the Organization of American States (OAS), Special Programme for Research and Training in Tropical Diseases (TDR)/ World Health Organization (WHO), International Foundation for Science (IFS), Fundación Natura, and Secretaria Nacional de Ciencia, Tecnología e Innovación (SENACYT) of Panama for financial support. Thanks are also due to the National Environmental Authority of Panama for granting permission to collect plants in national parks, Alex Espinosa, Carlos Guerra and Mireya Correa for the taxonomic identification of the plants. JS-W is grateful to Secretaria Nacional de Ciencia, Tecnología e Innovación (SENACYT), Panama for the Ph.D. scholarship.
Declaration of interest
The author declare no conflicts of interest.
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