Triterpenes from Minquartia guianensis (Olacaceae) and in vitro antimalarial activity

ARTIGO

Triterpenes from Minquartia guianensis (Olacaceae) and in vitro antimalarial activity^#

Lorena Mayara de Carvalho Cursino^I; Cecilia Veronica Nunez^I,*; Renata Cristina de Paula^II; Maria Fernanda Alves do Nascimento^II; Pierre Alexandre dos Santos^III

^ILaboratório de Bioprospecção e Biotecnologia, Instituto Nacional de Pesquisas da Amazônia, Av. André Araujo, 2936, 69060-001 Manaus - AM, Brasil

^IIDepartamento de Produtos Farmacêuticos, Faculdade de Farmácia, Universidade Federal de Minas Gerais, Av. Pres. Antonio Carlos, 6627, 31270-901 Belo Horizonte - MG, Brasil

^IIIFaculdade de Ciências Farmacêuticas, Universidade Federal do Amazonas, Rua Alexandre Amorim, 330, 69010-300 Manaus - AM, Brasil

ABSTRACT

Minquartia guianensis, popularly known as acariquara, was phytochemically investigated. The following triterpenes were isolated from the dichloromethane extract of leaves: lupen-3-one (1), taraxer-3-one (2) and oleanolic acid (3). The dichloromethane extract of branches yielded the triterpene 3β-methoxy-lup-20(29)-ene (4). The chemical structures were characterized by NMR data. Plant extracts, substance 3, squalene (5) and taraxerol (6), (5 and 6 previously isolated), were evaluated by in vitro assay against chloroquine resistant Plasmodium falciparum. The dichloromethane extract of leaves and the three triterpenes assayed have shown partial activity. Thus, these results demonstrated that new potential antimalarial natural products can be found even in partially active extracts.

Keywords:Minquartia guianensis; triterpenes; Plasmodium falciparum.

INTRODUCTION

Minquartia guianensis Aubl. popularly known as acariquara, acari, arariúba, among others,¹ occurs in the Amazon region, Nicaragua, Panama and Costa Rica.² It belongs to the Olacaceae which has 24 genus and 150 species.³ From this family, several chemical classes have been isolated such as alkaloids,^4-9 proanthocyanidins,^10,11 chromones,¹² steroids,¹³ flavonoids,^12,14 isoprenoids,¹² polyisoprenoids,¹⁵ diterpenes,^16,17 sesquiterpenes,⁹ triterpenes,^10,13,18-20 and glycerol-derivatives.¹²

Previous phytochemical studies on M. guianensis reported the isolation of triterpenes,^18,20 xanthone and minquartynoic acid.^20,21 The minquartynoic acid isolated from bark chloroform extract showed activity against Plasmodium falciparum and Leishmania major.²²

This paper describes the isolation and identification of four triterpenes which have been isolated from dichloromethane extracts of leaves and branches as well as reporting the antimalarial activity evaluation of the plant extracts and three triterpenes, two of which were previously isolated from this species.¹⁸

RESULTS AND DISCUSSION

Chemistry

Fractionation of leaf and branch extracts yielded four known triterpenes. Triterpenes 1-3 were purified from the dichloromethane extract of leaves while triterpene 4 was isolated from the dichloromethane extract of branches (Figure 1).The identifications were carried out by ¹H and ¹³C (APT and DEPT) NMR spectral data analysis, including 2-D NMR experiments (COSY, HSQC and HMBC).

Compounds 1 and 2 have shown yellow spots in TLC with anisaldehyde sulphuric acid reagent. The ¹H-NMR spectrum exhibited seven more intense signals between 0.7 and 1.7 ppm suggesting the presence of triterpene. Two intense signals were observed at δ_H 4.55 (1H, dd, J = 2.5 and 1.0 Hz) and 4.67 (1H, d, J = 2.5 Hz) indicating the presence of the lupane skeleton. Another signal with low intensity was observed at δ_H 5.54 (1H, dd, J = 8.0 and 3.0 Hz) indicating signals of the taraxerane skeleton.

The composition of the triterpene mixture was determined by the difference in intensity of signals by ¹³C-NMR data spectra analysis.^23,24 The ¹³C-NMR spectrum showed two intense signals at δ_C 151.0 and δ_C 109.6, corresponding to a quaternary carbon and a methylene carbon of the double bond, respectively, confirmed by DEPT spectra. Another signal at δ_C 218.3 was attributed to a carbonyl group. These signals confirmed the presence of the lupane skeleton, with a carbonyl group at position 3. The ¹³C-NMR spectra also showed two less intense signals at δ_C 157.8 and 117.4, corresponding to a quaternary carbon and a methyne carbon of the taraxerane double bond, respectively, also confirmed by DEPT spectra.

Other data were compared with those available in literature^25,26 and allowed the identification of 1 and 2 as the two triterpenes lupen-3-one and taraxer-3-one, respectively (Table 1). This is the first report of taraxer-3-one occurrence in Olacaceae while lupen-3-one was only previously identified in Olacaceae from leaves of M. guianensis.¹⁸

The ¹H-NMR spectrum of compound 3 showed several signals in the shielding region between δ_H 0.7 and δ_H 1.4. The deshielding region revealed a signal at δ_H 5.30 (1H, t, J = 4.0 Hz), which together with a signal at δ_H 2.82 (1H, dd, J = 14.0 and 4.0 Hz) indicated the oleanane skeleton. The ¹³C-NMR spectral data exhibited signals at δ_C 122.8 and 143.8 corresponding to the carbons in C-12 and C-13, respectively. The signal at δ_C 173.9 was assigned to the carboxyl group (C-28). Other signals were compared with literature data (Table 1).^27,28 Taken together, this data allowed the identification of compound 3 as oleanolic acid which, to the best of our knowledge, is described here for the first time in Olacaceae.

Compound 4 showed a purple color in TLC when revealed with anisaldehyde sulphuric acid reagent. The ¹H-NMR spectrum exhibited a doublet at δ_H 4.69 (1H, J = 2.0 Hz) and a doublet of doublet at δ_H 4.57 (1H, J = 2.0 and 1.0 Hz). At δ_H 3.35 (3H, s) a signal of hydrogen of a methoxy group which is linked in C-3 was observed, as confirmed by HMBC. Signals at δ_H 2.63 (1H, dd, J = 12.0 and 4.0 Hz) revealed protection of the H-3. The ¹³C-NMR spectrum showed signals at δ_C 150.9 and δ_C 109.3, corresponding to a quaternary carbon (C-20) and a methylene carbon (C-29), respectively, by DEPT, suggesting the presence of a lupane skeleton.²⁷ The carbinol signal was observed at δ_C 88.7 and the methoxy group appeared at δ_C 57.5. The HSQC experiment showed the couplings of δ_C 109.3 (C-29) to δ_H 4.57 (H-29), δ_C 109.3 (C-29) to δ_H 4.69 (H-29), δ_C 88.7 (C-3) to δ_H 2.63 (H-3) and δ_C 57.5 (C-31) to δ_H 3.35 (H-31). In the HMBC spectrum, the following correlations, among others, were observed: signal at δ_C 88.7 to δ_H 0.95 (³J), δ_C 88.7 to δ_H 3.35 (³J), δ_C 48.3 to δ_H 4.69 (³J), δ_C 19.3 to δ_H 4.69 (³J) and δ_C 48.3 to δ_H 4.57(³J). Assignment of H-29 (δ_H 4.57) and H-30 (δ_H 1.68) was determined by ¹H-¹H COSY spectrum analysis.

As the ¹³C-NMR data of 4 are not available in the literature consulted, we performed a calculation in order to estimate the chemical shifts of 4 by subtracting the effect of the OH group in C-3 and adding the OCH₃ group in its place, and then calculating the α, β and γ effects on the carbon chemical shift (Table 2). Lupeol is a common triterpene with this skeleton and hence the Dδ_C calculation was based on its ¹³C-NMR data.²⁹ These calculations allowed the identification of 3β-methoxy-lup-20(29)-ene which is reported for the first time in Olacaceae.

Antiplasmodial activity

Dichloromethane extracts of leaves and branches from M. guianensis, oleanolic acid (3), squalene (5) and taraxerol (6) (Figure 2) (previously isolated)¹⁸ were assayed in two concentrations (50 and 25 µg/mL) against P. falciparum (W2) and were found to be partially active except for dichloromethane extract from branches (Table 3).

Compound 3, isolated here from dichloromethane extract of leaves, caused 51% parasitemia reduction against P. falciparum (W2), at 50 µg/mL concentration and 28% reduction at 25 µg/mL. The two compounds previously isolated, squalene (5) and taraxerol (6), also from the dichloromethane extract of leaves,¹⁸ showed slightly better activity, with 67 and 32% reduction for squalene and 64 and 34% for taraxerol, at 50 and 25 µg/mL, respectively. These results allowed prediction of the IC₅₀ range of 25-50 µg/mL for the assayed triterpenes 3, 5 and 6. However, the IC₅₀ could not be determined because of the small amount of the triterpenes. IC₅₀ was determined only for the positive control, chloroquine (Figure 3). In the literature available, only compound 3 was assayed against Plasmodium falciparum, and exhibited IC₅₀ values of 88.8 and 70.6 mg/mL against chloroquine resistant (K1) and sensitive (T9-96) P. falciparum, respectively.³⁰ Our results revealed that 3 is even more active against the chloroquine resistant P. falciparum W2 clone. According to Sairafianpour and collaborators, the antiplasmodial activity of oleanolic acid might be due to its incorporation into the erythrocyte membrane, which would affect Plasmodium growth.³¹

EXPERIMENTAL

General

The compounds isolated were characterized by their NMR spectra which were recorded on a Varian spectrometer operating at 500 MHz (¹H) and at 125 MHz (¹³C) using CDCl₃ as the sample solvent and internal standard. High performance liquid chromatography (HPLC-DAD) was carried out with analytic and semi-preparative cyanopropyl columns (Luna-Phenomenex^®). The fractionations were carried out by the classical techniques including open chromatographic column and comparative thin layer chromatography (TLC) of silica gel 60 (230-400 mesh, ASTM, Merck) and Florisil (100-200 mesh, ASTM, Merck) to guide the fractions analysis.

Plant material

Minquartia guianensis Aubl. leaves and branches were collected at the Reserva Ducke, Manaus, Amazonas, Brazil, in April 2005. A plant voucher specimen was compared with one previously deposited (179.806) in the herbarium of the National Institute of Amazonian Research - INPA, Manaus, AM, Brazil.

Extraction and isolation

M. guianensis dry leaves (289 g) and branches (281 g) were powdered and extracted three times with CH₂Cl₂, using an ultrasound bath (20 min) each time, yielding the crude extracts (3.5 and 8.5 g, respectively).

The dichloromethane extract of leaves was fractionated on a chromatographic column using silica gel 60 as the stationary phase, and eluted with solvents of increasing polarity (hexane, AcOEt and MeOH). From this column, 2 fractions were re-fractionated (fractions 10-13 and 23-29). Fraction 10-13 (425 mg) was rechromatographed on a chromatographic column using Florisil as a stationary phase, and eluted with gradients of hexane, AcOEt and MeOH; subfraction 4 yielded the mixture of compounds 1 and 2 (2.3 mg) by recrystallization. Fraction 23-29 (480 mg) was submitted to a chromatographic column using silica gel 60 and eluted with a gradient of CH₂Cl₂ and acetone. Subfractions 9-11 (25 mg) were purified by HPLC eluted with acetonitrile (isocratic) on a cyanopropyl column (Phenomenex^®), 250 x 10 mm, 5 µm with 25 µL of injection volume, yielding compound 3 (2 mg) (Rt = 40.2 min).

The dichloromethane extract of branches was fractionated on a chromatographic column using silica gel 60 as the stationary phase, and eluted by a gradient of hexane, AcOEt and MeOH. Fraction 3-5 (74.8 mg) was rechromatographed on silica gel 60 using a gradient of hexane, CH₂Cl₂ and AcOEt. Sub-fractions 8-14 (27 mg) were recrystallized yielding compound 4 (3.2 mg).

Plasmodium falciparum continuous culture and in vitro assay

The P. falciparum W2 clone was kept in a continuous culture at 37 ºC on human erythrocytes using the candle jar method in RPMI medium supplemented with 10% human plasma (complete medium), as previously described.³² Synchronization of the parasites was achieved by sorbitol treatment³³ and parasitemias were determined microscopically in Giemsa-stained smears.

In vitro assays were carried out with erythrocytic cultures of chloroquine resistant Plasmodium falciparum (W2 clone), by the HRP2 method.³⁴ Briefly, the assay was started by parasite-drug incubation: 20 µL of each extract or substance diluted (stock sample concentration: 50 µg/mL, in DMSO) was placed in 96-well microplates in duplicate, which contained 180 µL of infected erythrocyte suspension (1.5% hematocrit, 0.05% parasitemia). Controls without drugs were used, with infected erythrocytes (positive control) or infected erythrocyte, frozen 24 h after starting the assay (background). The microplates were then incubated in a 5% CO₂ atmosphere at 37 ºC for 48 h. After this period, the microplates were frozen (-20 ºC for at least 24 h), in order to promote erythrocyte lysis. The cell lysis content (100 µL/well) was transferred to 96-well plates, previously incubated overnight with anti-HRP2 capture antibody (MPFM-55A ICLLAB^®) and blocked with 200 µL/well of blocking solution (PBS/BSA 2%). After 1 h of incubation, the plates were washed and incubated with 100 µL/well of the peroxidase-conjugated secondary antibody for a further 1 h (MPFG55P-ICLLAB^®). The plates were again washed and incubated for 10 min in the presence of TMB substrate solution. The reaction was stopped with HCl 1 N and the absorbance measured on a spectrophotometer (450 nm). The percentage reduction of parasite growth was calculated from the absorbance measure.

ACKNOWLEDGEMENTS

The authors thank CT-Agro/CNPq/MCTI (520281/2007-1 and 562892/2010-9) and PPBio/CNPq/MCTI (558321/2009-7) for the fractionation financial support, to the Phytochemistry Laboratory of Faculty of Pharmacy/UFMG and the Rede de Produtos Naturais para Quimioterapia Antimalárica (Natural Products Net for Antimalarial Chemotherapy), PRONEX/CNPq (555655/2009-1) and FAPEMIG (CDS APQ 01129-10), coordinated by Profa. Dra. A. B. de Oliveira, for the in vitro assays financial support.

Recebido em 24/5/12; aceito em 21/9/12; publicado na web em 9/11/12

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Artigo em homenagem ao Prof. Otto R. Gottlieb (31/8/1920-19/6/2011)

Publication Dates

• Publication in this collection
  30 Nov 2012
• Date of issue
  2012

History

• Received
  24 May 2012
• Accepted
  21 Sept 2012