ARTÍCULO ORIGINAL

 

In vitro antimalarial activity of fractions and constituents isolated from Tabebuia billbergii

 

Actividad antimalárica in vitro de fracciones y constituyentes aislados de Tabebuia billbergii

 

 

MSc. Harold Gómez-Estrada,^I PhD. Ricardo Gaitán-Ibarra,^I MSc. Fredyc Díaz-Castillo,^I PhD. Hilda A. Pérez,^II PhD. José D. Medina^III

^I Universidad de Cartagena. Cartagena-Colombia.
^II Instituto Venezolano de Investigaciones Científicas. Venezuela.
^III Universidad Central de Venezuela. Caracas, Venezuela.

 

 

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ABSTRACT

Introduction: in vitro antimalarial activity of naphthoquinones (1-5), isolated from Tabebuia billbergii (Bureau & K. Schum.) Standl., was investigated. Tabebuia billbergii, commonly known as guayacán, is a plant traditionally used in the Amazon in numerous conditions like bacterial and fungal infections, fever, syphilis, malaria, trypanosomiasis, as well as stomach and bladder disorders, and tumours.
Objective: to study the dichloromethane extracts of both the trunk and the inner bark of Tabebuia billbergii and to demonstrate the antimalarial activity of some of its bioactive components.
Methods: some bioactive components were evaluated for the antimalarial activity against Plasmodium berghei, by using the inhibition of the differentiation cycle of the parasite measure by the ³H-hypoxanthine incorporation and compared to that obtained for chloroquine.
Results: conventional chromatographic techniques and bioassay-guided fractionation (Artemia salina) allowed isolating from the active fractions one naphthoquinone (lapachol) and four naphtho-furan-4,9-diones. These compounds proved to have an important antiplasmodial effect, with very encouraging IC₅₀'s, especially when compared to the results shown by Chloroquine in the same experiment. In addition, two triterpenes, β-sitosterol and stigmasterol, were obtained from the bark.
Conclusions: the activity-guided fractionation (A. salina) of dichloromethane extracts of the trunk and the inner bark of Tabebuia billbergii led to the isolation and the identification of five quinonoid compounds with antiplasmodial effect. The significant inhibitory activity in vitro against Plasmodium berghei observed for compound 2-acetyl-naphtho-[2,3b]-furan-4,9-dione allow us to present them as a potential antimalarial compound.

Key words: Tabebuia billbergii, naphthoquinones, Plasmodium berghei.

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RESUMEN

Introducción: se evaluó la actividad antimalárica in vitro de una serie de naftoquinonas (1-5), aisladas de Tabebuia billbergii (Bureau & K. Schum.) Standl., que es conocida comúnmente como guayacán, una planta utilizada tradicionalmente en la Amazonía en numerosos problemas de salud como infecciones bacterianas y fúngicas, fiebre, sífilis, paludismo, tripanosomiasis, así como en problemas estomacales, tumores y trastornos de la vejiga.
Objetivo: estudiar los extractos en diclorometano tanto del tronco como la corteza interna de Tabebuia billbergii y evaluar la actividad antimalárica de algunos de sus componentes bioactivos.
Métodos: la actividad antimalárica contra Plasmodium berghei se evaluó en algunos componentes bioactivos, por la inhibición del ciclo de la diferenciación de la medida de los parásitos mediante la incorporación de 3H-hipoxantina y se comparó con la obtenida para la cloroquina.
Resultados: a través de técnicas cromatográficas convencionales y el fraccionamiento guiado por bioensayo (Artemia salina) se aislaron de las fracciones activas, una naftoquinona (lapachol) y 4 nafto-furan-4,9-dionas. Estos compuestos presentaron un efecto antiplasmodial importante, con buenos valores de IC₅₀, especialmente cuando se compara con los resultados mostrados por la cloroquina en el mismo experimento. Además, se obtuvieron de la corteza 2 triterpenos, β-sitosterol y estigmasterol.
Conclusiones: el fraccionamiento guiado por Artemia salina de los extractos en diclorometano del tronco y la corteza interna de Tabebuia billbergii, condujo al aislamiento y la identificación de 5 compuestos de naturaleza quinoidal con efecto antiplasmódicol. La actividad in vitro contra Plasmodium berghei observada para el compuesto 2-acetil-nafto-[2,3 b]-furan-4,9-diona, permite proponerlo como un potencial compuesto antimalárico.

Palabras clave: Tabebuia billbergii, naftoquinonas, Plasmodium berghei.

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INTRODUCTION

Malaria is a serious infectious disease caused by protozoan parasites in tropical and subtropical regions. Global spread of multiple drug-resistant malaria has become a major health problem and efforts to search for new antimalarial are needed. The inner bark of Tabebuia spp. has been used for many years by the Tukuna Indians of the Colombian Amazon as antimalarial.^1-3 The genus Tabebuia belongs to the Bignoniaceae family. It comprises about 100 species that are distributed from Mexico to northern Argentina. Many of these species have interesting biological activities against tumours, viruses, bacteria and the plasmodia.⁴ Previous studies on the chemistry of some Tabebuia spp have resulted in the isolation of naphthoquinone and anthraquinone derivatives,^5,6 which were shown active against malaria,^7-9 bacteria¹⁰ as well as cancer cells.^11,12 A particularly interesting species is Tabebuia chrysantha, from which lapachol and â-lapachone were isolated, together with several other naphthoquinones and antraquinones, which have shown antitumoral activity.^13-15 From T. cassinoides were isolated three naphtho-furan-diones, which were shown to have in vitro cytotoxic activity against KB cells.¹⁶ Natural and synthetic naphthoquinones have shown significant antimalarial activity against chloroquine-resistant Plasmodium parasites.^17,18

These important findings justified our studies on the flora of the Atlantic coast of Colombia. As a result, this paper deals with the isolation of lapachol and several naphtho-furan-4,9-diones from the trunk and inner bark of Tabebuia billbergii (Bureau & K. Schum.) Standl. (Bignoniaceae) and their effects on the differentiation cycle of Plasmodium berghei, measured by the ³H-hypoxanthine incorporation.

 

METHODS

General

Column chromatography (CC): silica gel 60 Merck (70-230 mesh). Preparative TLC: pre-coated TLC plates, silica gel 60 F₂₅₄ (2 mm, Merck). UV: spectrophotometer Milton Roy Spectronic 3000 Array; 1mg/mL MeOH. FT-IR spectra: (KBr) spectrophotometer Nicolet 5DX. ¹H-NMR spectra: Bruker AM 300 (300 MHz) in CDCl₃, TMS as internal standard. EIMS: Kratos MS25RFA, at 70 eV, in brackets identification and relative abundance. GC-MS: Hewlett-Packard GC-5890 Series II, with a Mass detector and a Wiley library of spectra, DB-5, 30 m column and He as carrier gas.

Plant Material

In April of 2008, samples from the trunk and inner bark of T. billbergii were collected in the vicinity of Arjona, located in the Department of Bolívar, Colombia. The species was taxonomically identified by Hermes Cuadros (Universidad del Atlántico, Barranquilla-Colombia) and a voucher specimen Nº 258, deposited in the Botanical Garden "Guillermo Piñeres", Cartagena, Colombia. The name was cross-referenced with the International Plant Name Index (IPNI) and W3Tropicos.

Extraction

Powdered dry wood (5 kg) and inner bark (4 kg) were successively introduced into a Soxhlet apparatus with hexane, dichloromethane and ethanol. Evaporation of the solvents under reduced pressure produced the following residues: 2.5, 6.1, and 15.8 g from the wood and 2.8, 8.5, and 12.8 g from the bark. These extracts were evaluated through lethality tests against Artemia salina,¹⁹ in order to select the more active ones for subsequent fractionation.

Fractionation of the dichloromethane trunk wood extract

Column chromatography (CC) of the CH₂Cl₂ extract of the trunk wood using eluents of increasing polarity from hexane to AcOEt-MeOH (1:1), produced 150 fractions (20 mL ea.) which were monitored by TLC and combined according to their TLC profiles to obtain 9 fractions. At this stage, all the fractions were assayed against A. salina. Fractions 2 and 4 showed the best LC₅₀'s and were further fractionated. Fraction 2 (280 mg) was chromatographed by column using hexane with increasing amounts of AcOEt as eluent. The fraction obtained from hexane-AcOEt (1:1), was further purified by PTLC (CH₂Cl₂) and yielded compound I (Fig.). Fraction 4 (80 mg) was subjected to PTLC (Hexane-AcOEt, 1:1) and yielded compound II (Fig.).

Fractionation of the dichloromethane inner bark extract

Column chromatography of the CH₂Cl₂ extract of the inner bark, under the same conditions mentioned above, produced 220 fractions (20 mL ea.). They were monitored by TLC and combined to obtain 17 fractions according to their TLC profiles. Of these, fractions 3, 5, 6, 12 and 17 showed good LC₅₀'s and were assessed against Plasmodium berghei. These active fractions were further fractionated. Fraction 3 was purified by repeated PTLC to yield compound IV (Fig.). Fraction 5 purified by PTLC with Hexane-CH₂Cl₂ (3:7) produced compounds II and III (Fig.). Fraction 6 treated in the same manner produced compound 3, together with β-sitosterol and stigmasterol. Fraction 12 subjected to PTLC with CH₂Cl₂ produced compound V (Fig.).

Compounds 1-5 were identified using spectral data such as ¹H NMR, ¹³C NMR, as well as EI-MS spectra, and by direct comparison with the data reported in the literature, as lapachol (I), 2-acetyl-8-methoxy-naphtho-[2,3b]-furan-4,9-dione (II),²⁰ 2-acetyl-7,8-dimethoxy-naphtho-[2,3b]-furan-4,9-dione (III),²¹ 2-acetyl-naphtho-[2,3b]-furan-4,9-dione (IV),^5,9 and 2-acetyl-7-hydroxi-8-methoxy-naphtho-[2,3b]-furan-4,9-dione (V)⁶ (Fig.). β-sitosterol and stigmasterol were separated by GC-MS and identified through comparison with their spectra in the instrument's library.

Artemia salina lethality tests

These tests were performed using the protocol reported in the literature.^19,22 Briefly, a few milligrams of brine shrimp eggs were placed for hatching in seawater. After 10-12 h, the eggs began hatching. Two days were allowed for the shrimp to mature into nauplii (shrimp can be used 48-72 h after the initiation of hatching). Stock solutions of extracts and fractions were prepared by dissolving extracts in saltwater. Working solutions of 10 to 1000 ppm were prepared by dilution and placed in culture tubes. Ten nauplii were placed into each tube. Tubes were incubated for 24 h at room temperature under illumination. Three replicates were prepared for each concentration. After 24h, the numbers of dead nauplii were counted. All results are expressed as mean LC₅₀ values ± standard deviation () of at least three independent experiments.

Antimalarial activity

Estimation of antimalarial activity was performed as reported in the literature.⁹ The biological activity of the fractions and isolated compounds was assessed in Plasmodium berghei using the inhibition of the differentiation cycle of the parasite measured through the ³H-hypoxanthine incorporation. Short- term cultures of Plasmodium berghei followed the protocol described by Kamiyama and Matsubara (1992).²³ Chloroquine was used as control.

Statistical Analyses

The lethal concentration 50 of A. salina (LC₅₀) and the inhibitory concentration 50 % (IC₅₀) of Plasmodium berghei were calculated on the basis of a nonlinear regression (curve fit), Statistical analyses were performed by one-way analysis of variance and Newman»Keuls multiple comparison tests. Differences were considered significant where p values were < 0.05.²⁴

 

RESULTS

The extracts from the trunk and inner bark of T. billbergii were evaluated by conducting lethality tests against Artemia salina¹⁹ and selecting the more active extracts for fractionation. In both cases, the CH₂Cl₂ extracts showed to be the more actives, giving lethal concentrations (LC₅₀) at 163 and 58 mg/mL, respectively (Table 1).

Fractionation of these extracts produced several active fractions (Table 2) which were assessed for antiplasmodial activity against Plasmodium berghei (Table 3). Posterior separation permitted the isolation and identification of the quinonic compounds 1-5, together with the triterpenoids β-sitosterol and stigmasterol.

Compounds 1-5 were identified using spectral information such as that from ¹H NMR, ¹³C NMR, as well as EI-MS spectra, and by direct comparison with the data reported in the literature, as 2-hydroxy-3-(3^'methyl-2^'-butenyl)-naphthalen-1,4-dione (lapachol, 1), 2-acetyl-8-methoxy-naphtho-[2,3b]-furan-4,9-dione (2),²⁰ 2-acetyl-7,8- dimethoxy-naphtho-[2,3b]-furan-4,9-dione (3),²¹ 2-acetyl-naphtho-[2,3b]-furan-4,9-dione (4)^5,9 and 2-acetyl-7-hydroxi-8-methoxy-naphtho-[2,3b]-furan-4,9 -dione (5).⁶ β-sitosterol and stigmasterol were separated by GC-MS and identified by comparison with their spectra in the instrument's library.

The naphtho-furan-diones showed important inhibitory activity when assayed in vitro against Plasmodium berghei, especially in comparison to the activity exhibited by Chloroquine which was used as control. The IC₅₀ values obtained with these pure compounds are summarized in Table 3.

 

DISCUSSION

The activity-guided fractionation (A. salina) of different extracts from the wood and inner bark of T. billbergii led to the isolation and identification of five quinonoid compounds. The results from the spectral data for compounds I-V isolated in this study were compared with the data reported previously in the literature for these compounds. The antimalarial activity of the fractions and isolated compounds was assayed against Plasmodium berghei by using the inhibition of the differentiation cycle of the parasite measured through the ³H-hypoxanthine incorporation. Short-term cultures of Plasmodium berghei followed the protocol described by Kamiyama and Matsubara (1992).²³ Chloroquine was used as control for the experiments. The IC₅₀ values obtained with various fractions and isolates from the plant are summarized in Tables 1 and 2.

Preliminary evaluation of the antiparasitic activity of compounds 1, 2, 3 and 5, suggests there is much ground for further investigation, since the values of IC₅₀'s obtained are rather encouraging. The activity observed for compound 4 deserves special attention because of its pharmaceutical potential. Of course, this requires further deep research, but we are already involved in the synthesis of some of these quinones, in order to be able to proceed with the in vivo tests.

 

ACKNOWLEDGEMENTS

This work was partially financed by Colciencias, Colombia, and by the Instituto Venezolano de Investigaciones Científicas (IVIC). We are grateful to Prof. Hermes Cuadros, Universidad del Atlántico, Barranquilla, Colombia, and Prof. Prisciliano Martínez, Facultad de Ciencias Químicas Farmacéuticas, Universidad de Cartagena, for the collection and identification of plant material. HGE acknowledges Colciencias and the Universidad de Cartagena for a graduate study fellowship. JDM is grateful to Dr. Werner Wilbert, of the Department of Anthropology of the Instituto Venezolano de Investigaciones Científicas (IVIC), for his kind review of the manuscript.

 

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Recibido: 1 de diciembre de 2011.
Aprobado: 30 de enero de 2012.

 

 

Harold Gómez-Estrada. Facultad de Ciencias Farmacéuticas, Universidad de Cartagena. Grupo de Investigación en Química de Medicamentos (GIQM). Cartagena, Colombia. Teléf.: 57-5-6698323. Correo electrónico: [e-mail]