Antiprotozoal activity of (8-hydroxymethylen)-trieicosanyl acetate isolated from Senna villosa.

Abstract

A white solid compound was isolated from the chloroform extract of the leaves of Senna villosa. The material was identified by (1) H-NMR, [.sup.13] C-NMR, IR and EM methods as (8-hydroxymethylen)-trieicosanyl acetate, a new compound with biological activity, which was tested in vitro at concentrations of 1.65, 3.3 and 6.6 [micro].g/ml for inhibition of the growth of Trypanosoma cruzi epimastigotes and tripomastigotes. We observed inhibition of growth at all concentrations tested, and the effect at concentrations of 3.3 and 6.6 [micro]g/ml was greater than that of gentian violet (positive control). At the concentration of 6.6 [micro]g/ml, the compound showed the greatest inhibitory effect against the growth of both forms of the parasite. [c] 2008 Elsevier GmbH. All rights reserved.

Keywords: (8-hydroxymethylen)-trieicosanyl acetate; Senna villosa; Antiprotozoal activity

Introduction

Trypanosoma cruzi is the causative agent of Chagas' disease, which afflicts many in the poorest countries in the world. It is estimated that there are 16-19 million infected persons in South and Central America (WHO, 1995). This disease is the cause of about 50,000 deaths per year.

At present, compounds like allopurinol and gentian violet are used to treat Chagas' disease; however, they have several toxic side-effects. Traditional Mexican medicine uses a great variety of plants to combat this disease, one of which is Senna villosa Mill (Leguminoceae), commonly known as Salche, which grows in the Yucatan Peninsula of Mexico. In the traditional medicine of the ancient Mayas, the leaves of S. villosa were used to treat unidentified skin infections, dysmenorrhea (BADEPY, 1985), and inflammatory problems.

A preliminary phytochemical analysis of the leaves of S. villosa revealed quinones, sterols, flavonoids, and two 9,10-anthraquinones (Mena et al., 1997). Recently, the chloroform extract of the plant was tested against epimastigote and trypomastigote forms of Trypanosoma cruzi, and exhibited significant activity against both forms of the parasite (Guzman et al., 2004). This work describes the structure of (8-hydroxymethylen)-trieicosanyl acetate, isolated from the chloroform extract of S. villosa, and its antiprotozoal activity against T. cruzi. Some similar aliphatic hydroxy and oxo compounds have been isolated from plants. For example, pentaeicosanol-1 and 1-hydroxy-3-doeicosanone-2, showing antibacterial activity, were isolated from Heliotropium sinuatum (Modak et al., 2004), and ethyl 14-oxotetra-cosanoate was isolated from Cryptocoryne spiralis (Gupta et al., 1983). It was found that some longchain saturated fatty acids have anticoccidial activity (Winterrowd et al., 1989). In addition, 15-methoxytri-cosanoic acid was isolated from the caribbean sponge Callyspongia fallax (Carballeira and Pagan, 2001).

Materials and methods

General

Melting points were measured using a Fisher-Johns apparatus and are uncorrected. IR spectra were recorded on a Perkin Elmer FT Paragon 1000 spectrometer, using a KBr pellet. The [.sup.1] H- and [.sup.13] C-NMR spectra were recorded at room temperature in CDC[I.sub.3] on a 400 MHz Varian Mercury instrument, and chemical shifts are expressed relative to those of CDC[I.sub.3] (7.26 ppm). The correlation spectroscopy (COSY), distortionless enhancement by polarization transfer (DEPT) and heteronuclear multiple-quantum correlation (HMQC) experiments were also performed on this instrument. The MS was obtained on a Jeol GC-Mate at 70eV. Optical rotation was measured in a 1-cm cell using a Perkin-Elmer model 141 polarimeter at 20 [degrees]C. Elemental analysis was performed by USAI Facultad de Quimica of Universidad Autonoma de Mexico. A Varian 3800 gas chromatograph was used for purity determination.

Plant material

Leaves of S. villosa were collected in the town of Molas, Yucatan State, Mexico. Dr. Salvador Flores-Guido authenticated the material and a voucher specimen of the plant (10284) was deposited at the herbarium of Universidad Autonoma de Yucatan (UADY). The leaves were dried at room temperature under shade.

Extraction and isolation of (8-hydroxymethylen)-trieicosanyl acetate

Dry and powdered leaves (500 g) were extracted with CHC[I.sub.3] (31). The solvent was evaporated and the residue was chromatographed on silica gel (70-230 mesh), and eluted with hexane, increasing the polarity with ethyl acetate. Ten fractions were obtained, and the biological activity of each was determined. The most active fraction produced a white solid (yield 2.7%), which was recrystallized with chloroform. The purity of the compound was confirmed by GC analysis. [.sup.1] H-NMR: [delta] (ppm) 0.88 (t, 3H, J = 8.0Hz), 1.253 (m, 38 H), 1.571 (s, 3 H), 1.612 (m, 1 H), 2.287 (t, 2 H, J = 7.6 Hz), 4.052 (t, 2 H, J = 6.4 Hz).

[.sup.13] C-NMR: [delta] (ppm) 14.04, 22.66, 24.99, 25.87, 28.57, 29.09, 29.27, 29.39, 29.45, 29.51, 29.61, 31.85, 34.34, 64.25, 173.65.

Trypanocidal in vitro activity

Biological activity was assayed using epimastigotes and trypomatigotes of T. cruzi strain Y isolated from human blood cultured in liver infusion Tryptose medium (LIT, Bacto) supplemented with 10% heat-inactivated fetal calf serum and cultured at 28 [degrees]C. Experiments were carried out in triplicate, using glass tubes containing 5 x [10.sup.6] protozoa/ml in a final volume of 3ml, which was homogeneous at the early log phase culture of epimastigotes or trypomastigotes (Fournet et al., 1994; Araujo et al., 1999; Muelas-Serrano et al., 2000). (8-hydroxymethylen)-trieicosanyl acetate was dissolved in phosphate-buffered saline (PBS) pH 7.6, passed through a Millipore 0.22-[micro]m pore size filter and diluted to concentrations of 1.65, 3.3 and 6.6[micro]g/ml. These solutions were placed into tubes for each experiment.

Gentian violet (17.5 mg/ml) was used as a positive control. Parasites were counted daily after contact with the compound using a Neubauer chamber, for 10 days. The results were compared with those of controls grown without drug (Osuna et al., 1990; Rojas de Arias et al., 1995; Castilla et al., 1996). A similar procedure was carried out to test cytotoxicity. Vero cell lines free of infection were strained with trypan Blue and results were expressed as percent of viability in relation to negative controls. Results are presented as mean values [+ or -] S.D.

Results and discussion

The chloroform extract of the leaves of S. villosa yielded a white solid compound ((8-hydroxymethylen)-trieicosanyl acetate) mp 76-77 [degrees]C, [[alpha]][D.sup.20] = -1.2 (c = 0.05, CHC[1.sub.3]). Anal Calc. C, 78.79; H, 13.13 found C, 78.70; H, 13.37. IR [v.sub.max] 3451, 1105 (hydroxyl groups), 1735 (ester CO) and 717[cm.sup.-1] (long chain). [['M].sup.+] at 412 suggesting the molecular formula as [C.sub.26][H.sub.52][O.sub.3]. Two characteristic fragment ions at m/z 43 and 57 indicated the presence of an acetate (Hesse et al., 1997). [alpha]-Fission ions at m/z 353, 339, 255, 211 and 201 indicated a hydroxymethylen group at C-8 (Gupta and Verma, 1990). The absence of an [[M-15].sup.+] indicates no branched methyl group, whereas the presence of an [[M + 1].sup.+] suggests and asymmetrical structure (Gupta et al., 1983).

The [.sup.1] H-NMR spectrum showed only an aliphatic region, indicating that it was a long-chain ester (Modak et al., 2004). The [.sup.1] H-NMR spectrum showed a triplet at [delta] 0.88, and in the HMQC spectrum it was correlated at [delta] 0.88 to C at [delta] 14.0, and was assigned to a terminal methyl group. A singlet at [delta] 1.57 correlated with C at [delta] 29.5, was assigned to an [alpha]-methyl group. A triplet was observed at [delta] 2.29, which integrated for 2 protons and was assigned to a [CH.sub.2] group adjacent to carboxyl [delta] 64.24). The multiplet at [delta] 1.25 integrated for 38 protons of several [CH.sub.2] groups. The multiplet at [delta]1.61 correlated to C at [delta] 28.57, which corresponded to CH. A triplet was observed at [delta] 4.05 and was assigned to a methylene group [alpha] to the hydroxy group ([delta] 64.25).

[FIGURE 1 OMITTED]

The connectivities of H-C (8) to [CH.sub.2] of hydroxymethylen and [CH.sub.2] (22) to [CH.sub.3] (23) were confirmed by the COSY data. The [.sup.13]C (DEPT)-NMR spectrum indicated that (8-hydroxymethylen)-trieicosanyl acetate contains [delta] 14.0 ([CH.sub.3]), 22.6 ([CH.sub.2]), 25.0 ([CH.sub.2]), 25.9 ([CH.sub.2]), 28.6 ([CH.sub.2]), 29.1 ([CH.sub.2]), 29.2 ([CH.sub.2]), 29.3 ([CH.sub.2]), 29.4 ([CH.sub.2]), 29.45 (CH), 29.5 ([CH.sub.3]), 29.6 ([CH.sub.2]), 31.8 ([CH.sub.2]), 34.3 ([CH.sub.2]), 64.24 ([CH.sub.2]), 64.25 ([CH.sub.2]).

[FIGURE 2 OMITTED]

The spectra data confirmed that the compound isolated from S. villosa is (8-hydroxymethylen)-trieico-sanyl acetate, which was tested against epimastigotes and tripomastigotes of T. cruzi, and the results are shown in Figs. 1 and 2.

When the compound was tested against epimastigotes, there was not a significant difference between the effects of the compound at 1.65, 3.3. and 6.6[micro]g/ml (Fig. I); however, the inhibition of growth with all concentrations was greater than that of gentian violet (positive control).

There was a significant difference between the effects of the compound against trypomastigotes (Fig. 2) at 1.65 [micro]g/ml and the other two concentrations; however, we observed inhibition of growth of trypomastigotes with all concentrations used. The effect of the compound at concentrations of 3.3 and 6.6[micro]g/ml is similar to that shown by gentian violet (positive control). At a concentration of 6.6[micro]g/ml, the compound showed the highest inhibitory effect against growth of both forms of the parasite, epimastigotes and trypomastigotes (Figs. 1 and 2), and reduced the number of epimastigotes by 92.6[+ or -] 0.4% and trypomastigotes by 89.7[+ or -]0.8%. Under the same conditions, gentian violet lysed 55[+ or -] 0.6% of the parasites.

Cytotoxicity in the Vero cell line was also tested with (8-hydroxymethylen)-trieicosanyl acetate; 96[+ or -] 2.0% of cell survival was observed at the highest concentration (6.6g/ml), which was very active against epimastigotes and trypomastigotes. This result was similar to that obtained with violet gentian (93 [+ or -] 3.0%).

These findings suggest that the (8-hydroxymethylen)-trieicosanyl acetate could be useful for the treatment of Chagas' disease, although it will be necessary to establish its mechanism of action.

References

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* Corresponding author. Tel.: + 525554837263; fax: + 525554837410.

E-mail address: [e-mail] (S.Perez).

0944-7113/$-see front matter [c] 2008 Elsevier GmbH. All rights reserved. doi: 10.1016/j.phymed.2008.02.013

E. Guzman (a), C. Perez (b), M.A. Zavala (b), K.Y. Acosta-Viana (a), S. Perez (b), *

(a) Departamento de Biomedicina de Enfermedades Infecciosas y Parasitarias, Centro de Investigaciones Regionales "Dr. Hideyo Noguchi", Universidad Autonoma de Yucatan, Av. Itzaez 490 x 59 Col. Centro. C.P. 97000, Merida, Yucatan. Mexico

(b) Departamento de Sistemas Biologicos, Universidad Autonoma Metropolitana-Xochimilco, Calzada del Hueso 1100, Col. Villa Quietud, A.P. 23-181, C.P. 04960, Mexico D.F., Mexico