Trypanocidal Activity of a New Diterpene from Casearia sylvestris var. lingua

• Abstract
• Materials and Methods
• References

Abstract

Bioassay-guided fractionation of the hexanic root bark extract of Casearia sylvestris var. lingua led to the isolation of a new clerodane diterpene, whose structure was elucidated as rel-(2S,5R,6R,8S,9S,10R,18S,19R)-19-acetoxy-18,19-epoxy-6-hydroxy-18-butanoyloxy-2-(2-methylbutanoyloxy)cleroda-3,13(16), 14-triene by spectroscopic means, including 1D and 2D NMR analyses. This compound showed pronounced activity on Trypanosoma cruzi, the casual agent of Chagas’ disease, with minimal inhibitory concentration (MIC) at 0.59 μg/mL.

Eighteen million people are estimated to be infected with Trypanosoma cruzi, the flagellate protozoan that causes Chagas’ disease. The two drugs used in clinical practice, nifurtimox and benznidazole, do not eliminate the parasites and resistance is reported [1]. In view of the need to find new drugs to treat Chagas’ disease, we have identified some plant species that presented a considerable toxic effect on T. cruzi. Among them, the hexanic root bark extract from C. sylvestris var. lingua displayed high trypanocidal activity with an MIC value of 9.53 μg/mL. It was fractionated by chromatography, and the fractions obtained were tested in the same anti-T. cruzi assay. The MIC values of the fractions were: 610.00 (10), 152.00 (1 - 3), 76.25 (4 and 5), 38.12 (6, 8 and 9), and 2.38 μg/mL (7). Fraction 7 was rechromatographed, and a diterpene 1 (Fig. [1]) was isolated. It was active on the parasite with an MIC value of 0.59 μg/mL. The toxic effect on T. cruzi of the hexanic root bark extract, the fractions, and the isolated substance were not transient, as the treated epimastigotes were not able to grow or move after their transfer to a fresh medium. In the control tests, the inactivity of dimethyl sulfoxide (DMSO) at the concentration used in the test was confirmed. Nifurtimox showed trypanocidal activity up to 19.06 μg/mL, thus the novel diterpene was 32 times more active than the reference drug tested.

The clerodane diterpenoid was isolated as a colorless viscous liquid. Its molecular formula was established to be C₃₁H₄₆O₈ by HR-FAB-MS and ¹³C-NMR spectral data suggesting nine degrees of unsaturation. The IR spectrum showed absorption bands at 1752, 1728, 1595 and 1458 cm^-1 indicating the presence of ester carbonyl moieties and ethylenic functionalities. The structure was further elucidated by examination of the ¹H-, ¹³C-, COSY, HSQC, HMBC and NOESY NMR spectra (Table [1]).

The ¹H-NMR spectrum showed characteristic signals for six methyl groups (δ = 0.88, 0.89, 0.9, 0.92, 1.14, 1.84), two acetal-acyloxy methine protons at δ = 6.45 (s) and at δ = 6.72 (t, J = 1.5 Hz), two oxymethines protons at δ = 3.8 (d, J = 7.4 Hz) and δ = 5.39 (d, J = 2.8 Hz) and an olefin proton at δ = 5.95 (dd, J = 1.5, 4.3 Hz). The presence of a terminal unsaturated methylene was suggested by the broad doublets at δ = 4.93, 5.01 and 5.12 (d, J = 17.4 Hz). The ¹³C-NMR spectrum contained 31 signals. The COSY 2D NMR spectrum showed peaks of correlation between H-10/H_{2 -} 1/H-2/H-3, H-6/H-7/H-8/H-17, H-11/H-12, H-14/H-15, H-2′/H-3′ and H-2″/H-3″, and vicinal coupling between H-2/H-3/H-18. Long-range correlations in the HMBC spectrum showed that the proton at δ = 2.29 attributed to H-10 correlated with C-1, C-2, C-5, C-6 and C-9, while the proton at δ = 3.8 (H-6) correlated with C-4, C-5, C-7, C-19. The proton H-3 correlated with C-1, C-2, C-5, C-6, and C-18 and H-8 correlated with C-7, C-9, C-11, C-17, C-20, thus confirming the basic skeleton of a clerodane diterpene [2]. Three esters moieties were identified by analysis of ¹³C-NMR data, and one of them was assigned as acetate ester (C = O and CH₃ at δ = 169.5 and 21.3, respectively). The 2-methylbutanoyloxy chain was located at C-2, based on the chemical shift of H-2 and on the HMBC correlations H-2/C-1′. The butanoyloxy group was placed at C-18 from a long-range correlation observed in the HMBC spectrum between H-18 and C-1″. The terminal unsaturated methylene group (H-15) correlated in the HMBC spectrum with C-13, C-14 and C-16, while H-12 correlated with C-9, C-11, C-13, and C-14 representing the six-carbon side chain attached to cycle A/B at C-9. The diacetal ring was constructed from the observation of a cross-correlation in the HMBC spectrum between the H-18 acetal proton with C-19, and the H-19 acetal proton with C-18. The relative stereochemistry was assigned based on the coupling constants and NOESY data. An equatorial position (α) was assigned for H-2 based on the J value of 3.0 Hz for the coupling between H-2 and H₂ - 1, thus the isobutyrate ester needs to be β axial. The absence of correlation between H-2 and H-10, an α axial proton, was noticed in the NOESY spectrum. A 1,3 diaxial NOE correlation was observed between H-8 and H-6, which constrained CH₃ - 17 to be equatorial like the C-6 hydroxy group (rel β). The acetal proton H-19 showed NOE correlations with H-18, H-10, and H₂ - 11 and was assigned a rel-α conformation. This required a substituent at C-5 to be rel-β leading to a cis A/B ring junction [2]. The NOE correlations observed between H-8 and CH₃ - 20; H-6 and H-8; H-18 and H-19; H-10 and CH₃ - 17 substantiated the positioning of H-10 and CH₃ - 17 on one side, and H-8 and CH₃ - 20 on the other. Thus, the active compound was identified as rel-(2S,5R, 6R,8S,9S,10R,18S,19R)-19-acetoxy-18,19-epoxy-6-hydroxy-18-butanoyloxy-2(2-methylbutanoyloxy)cleroda-3,13(16),14-triene. Compared with the clerodane diterpenes isolated from the genus Casearia, there was an absence of an acetoxy group in position C-18, but there was the presence of a butanoyloxy moiety in the diterpene [2], [3], [4], [5]. Its NMR spectral data were very similar to those reported for one compound isolated from Casearia tremula [2] and C. lucida [3] and identified as rel-(2S,5R,6R,8S,9S,10R,18S,19R)-18,19-diacetoxy-18,19-epoxy-6-hydroxy-2-(2-methylbutanoyloxy)-cleroda-3,13(16),14-triene. Copies of the original spectra are obtainable from the author of correspondence.

Although many diterpenes displaying pharmacological activities have been described in the genus Casearia [4], [5], [6], diterpenes of the variety lingua have never been identified or evaluated. Several natural substances, including diterpenes, of different plants have been shown to be active on T. cruzi [7], [8], [9], [10].

Materials and Methods

C. sylvestris Sw. var. lingua (Camb.) Eichl. (Flacourtiaceae) was collected in the Cerrado biome, in the surroundings of Brasília, Distrito Federal, Brazil, in 2002, and identified by Dr. José Elias de Paula, from the University of Brasília, and the voucher herbarium specimens were deposited in the Herbarium of said University, under the number J. Elias de Paula (UB) 3693.

Optical rotations were measured with a Perkin-Elmer 241 polarimeter at 20 °C. IR spectra were measured on a Bruker IR TF Tensor 27 spectrophotometer. The ¹H- and ¹³C-NMR spectra were recorded at 400.13 and 100.61 MHz, respectively, on a Bruker AMX-400 spectrometer at 300 K with a Bruker Gradient Unit (BGU) and an inverse gradient triple-resonance probe-head with a self-shielded gradient coil. The ¹H- and ¹³C-NMR chemical shifts are expressed in ppm relative to TMS, with coupling constants (J) given in Hz. The high-resolution mass spectra and FAB-MS were recorded on a JEOL MS700 apparatus.

Dried powdered root bark (400 g) of the C. sylvestris var. lingua was extracted with hexane (4 × 2 L). The extract (2.5 g) was chromatographed on a silica gel (230 - 400 mesh, 50 g, Ø column 4.5 cm, 10 mL fractions) eluting successively with toluene/ethyl acetate (1 : 0, 95 : 5, 90 : 10, 85 : 15, 80 : 20, 0 : 1, each 0.6 L) to give 10 fractions. Fraction 7 (eluted with toluene/ethyl acetate, 80 : 20, detection of eluates by TLC-SiO₂, toluene/ethyl acetate, 80 : 20; R_f: 0.2) (531 mg) was rechromatographed on a silica gel (32 g, Ø column 25 mm, 4 mL fractions), eluting with cyclohexane-ethyl acetate (75 : 25, 0.62 L) to furnish fourteen sub-fractions. The most abundant sub-fraction (eluted with cyclohexane/ethyl acetate, 75 : 25, detection of eluates by TLC-SiO₂, cyclohexane/ethyl acetate, 75 : 25; R_f: 0.13) (97 mg) was rechromatographed under the same conditions (silica gel 6 g, Ø column 13 mm, 4 mL fractions), which allowed the isolation of 92 mg of the active compound (eluted with cyclohexane/ethyl acetate, 75 : 25, detection of eluates by TLC-SiO₂, cyclohexane/ethyl acetate, 70 : 30; R_f: 0.21): Diterpene 1: viscous liquid; [α]²⁰ _D: + 26° (c 0.135, CH₂Cl₂); IR: ν_max = 1752, 1728, 1595, 1458 cm^-1. ¹H-NMR and ¹³C-NMR, see Table [1]. HR-FAB-MS: m/z = 569.3094 (calcd. for C₃₁H₄₆O₈Na: 569.3090).

Trypanosoma cruzi epimastigote forms were grown in a liver infusion tryptose medium (LIT) [11]. Parasites were dispatched in microtitre plates at 2 × 10⁶ cells/mL of LIT. Different concentrations of the crude extract, isolated fractions or the pure substance, previously diluted in DMSO (50 mg/mL) decreasing from 1.22 × 10³ down to 7.44 × 10^-2 μg/mL, were added to wells followed by incubation for 48 h. Activity was determined by evaluating the percentage of live parasites with a microscope. To confirm that the parasites were dead, the cells were washed and incubated for 72 h in LIT. The controls consisted of the incubation of parasites with DMSO (2 %), or the reference drug Nifurtimox [1] (Bayer). The bioassays were performed in triplicate.

References

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Prof. Dra. Laila Salmen Espindola

Laboratório de Farmacognosia

Faculdade de Ciências da Saúde

Universidade de Brasília

CEP

70910-900 Brasília - DF

Brasil

Phone: +55-61-307-2514

Fax: +55-61-273-0105

Email: darvenne@unb.br

References

• 1 Coura J R, de Castro S L. A critical review on Chagas disease chemotherapy.  Mem Inst Oswaldo Cruz. 2002;  97 3-24
  PubMedSearch in Google Scholar
• 2 Gibbons S, Gray A I, Waterman P G. Clerodane diterpenes from the bark of Casearia tremula .  Phytochemistry. 1996;  41 565-70
  CrossrefPubMedSearch in Google Scholar
• 3 Chaturvedula V, Prakash S, Hoch J M, Kingston D GI. Structure and stereochemistry of new cytotoxic clerodane diterpenoids from the bark of Casearia lucida from the Madagascar Rainforest.  J Nat Prod. 2002;  65 100-7
  CrossrefPubMedSearch in Google Scholar
• 4 Hunter M S, Corley D G, Carron C P, Rowold E, Kilpatrick B F, Durley R C. Four new clerodane diterpenes from the leaves of Casearia guianensis which inhibit the interaction of leukocyte function antigen 1 with intercellular adhesion molecule 1.  J Nat Prod. 1997;  60 894-9
  CrossrefPubMedSearch in Google Scholar
• 5 Oberlies N H, Burgess J P, Navarro H A, Pinos R E, Fairchild C R, Peterson R W. Novel bioactive clerodane diterpenoids from the leaves and twigs of Casearia sylvestris .  J Nat Prod. 2002;  65 95-7
  CrossrefPubMedSearch in Google Scholar
• 6 Morita H, Nakayama M, Kojima H, Takeya K, Itokawa M, Schenkel E P, Motidome M. Structures and cytotoxic activity relationship of casearins, new clerodane diterpenes from Casearia sylvestris Sw.  Chem Pharm Bull. 1991;  39 693-7
  CrossrefPubMedSearch in Google Scholar
• 7 Sepúlveda-Boza S, Cassels B K. Plant metabolites active against Trypanosoma cruzi .  Planta Medica. 1996;  62 98-105
  PubMedSearch in Google Scholar
• 8 Batista R, Chiari E, Oliveira A B. Trypanosomicidal kaurane diterpenes from Wedelia paludosa .  Planta Medica. 1999;  65 283-4
  Thieme ConnectPubMedSearch in Google Scholar
• 9 da Costa F B, Albuquerque S, Vichnewski W. Diterpenes and synthetic derivates from Viguiera aspillioides with trypanomicidal activity.  Planta Medica. 1996;  62 557-9
  PubMedSearch in Google Scholar
• 10 Alves T M, Chaves P P, Santos L M, Nagem T J, Murta S M, Ceravolo I P, Romanha A J, Zani C L. A diterpene from Mikania obtusata active on Trypanosoma cruzi .  Planta Medica. 1995;  61 85-7
  Thieme ConnectPubMedSearch in Google Scholar
• 11 Camargo E P. Growth and differentiation in Trypanosoma cruzi: I. Origin of metacyclic trypanosomes in liquid medium.  Rev Inst Med Trop de São Paulo. 1964;  6 93-100
  PubMedSearch in Google Scholar

Prof. Dra. Laila Salmen Espindola

Laboratório de Farmacognosia

Faculdade de Ciências da Saúde

Universidade de Brasília

CEP

70910-900 Brasília - DF

Brasil

Phone: +55-61-307-2514

Fax: +55-61-273-0105

Email: darvenne@unb.br