IDENTIFICATION OF A NEW AROMATIC GERANYL DERIVATIVE IN
THE RESINOUS EXUDATE OF HELIOTROPIUM FILIFOLIUM
(BORAGINACEAE)

ALEJANDRO URZÚA^*, BRENDA MODAK, RENE TORRES

Laboratory of Chemical Ecology, Faculty of Chemistry and Biology, University of Santiago of
Chile, Casilla 40, Correo 33, Santiago, Chile.
(Received: September 1, 2000 - Accepted: March 22, 2001)

ABSTRACT

A new aromatic geranyl derivative was isolated from the resinous exudate of Heliotropium filifolium. The structure was elucidated by high-resolution spectroscopic methods as filifolinyl senecionate. The compound has shown antibacterial activity against Escherichia coli K-12. After irradiation of a solution of filifolinyl senecionate with UV at 366 nm, the antibacterial activity was enhanced by 100%.

KEY WORDS: Heliotropium filifolium, Cochranea Section, Boraginaceae, resinous exudate, glandular trichomes, new aromatic geranyl derivative, Escherichia coli K-12, phototoxic compound.

RESUMEN

Del exudado resinoso de Heliotropium filifolium, se aisló un nuevo derivado aromático geranilado. Este compuesto se identificó por métodos espectrópicos de alta resolución como filifolinil senecionato. El compuesto mostró actividad antibacteriana contra Escherichia coli K-12. Esta actividad se vio incrementada en un 100% después de irradiar una solución de filifolinil senecionato con UV a 366 nm.

PALABRAS CLAVES: Heliotropium filifolium, Sección Cochranea, Boraginaceae, exudado resinoso, tricomas glandulares, derivado aromático geranilado, Escherichia coli K-12, compuesto fototóxico.

INTRODUCTION

Section Cochranea (Miers) Reiche of the genus Heliotropium Johnston (Boraginaceae), is known only from the coastal hills of northern Chile, and in the south of Perú. This section is of particular ecological interest, because the species grows in arid lands with extreme enviromental variation and conditions ¹⁾.

From the resinous exudate of H. filifolium, filifolinol (2), an aromatic geranyl derivative ²⁾ and flavonols ³⁾ were previously reported.

EXPERIMENTAL

Plant material

Heliotropium filifolium (Miers) Reiche (ST-2214 SSUC) was collected during the flowering season, October 1997, in the north of Vallenar (IV Region, Chile, 28 45'S, 70 49'W). Voucher specimens were deposited in the Herbarium of the Faculty of Biological Sciences of Catholic University of Chile, Santiago, Chile.

Extraction and isolation

The resinous exudate of H. filifolium was extracted by inmersion of the fresh plant material (900 g) in CH₂Cl₂, for 30 s at room temperature. The CH₂Cl₂ extract was concentrated to a sticky residue (220 g). Part of the extract (25 g) was purified by CC (silica gel) using a hexane-EtOAc step gradient.

Antibacterial activity determination

The quantitative antibacterial assay employed was an agar well diffusion assay ^4,5). Plates for the assay were prepared by dispersing 25 ml of sterile agar Muller-Hinton medium into 100 mm sterile Petri dishes. The solidified sterile agar plates were streaked uniformly with a dilution of the test organism by using the quadrant streak method. Wells with a diameter of 8 mm were produced using a sterile steel cylinder and the sample solution (1 mg/1ml in DMSO) was then added to each cylinder. Control plates with DMSO alone were done. A sample of harmane was used as standard control antibacterial agent for the assay, and as a well known phototoxic compound ⁶⁾.

Half of the plates were kept in the darkness, and the other half were irradiated with UV (366 nm) during 5 h.

The plates were incubated at 37 C for 24 h. Experiments were done in octuplicate. The average diameter of the inhibition zone is recorded in Table 3.

Spectroscopy

Both ¹H-NMR and ¹³C-NMR experiments were recorded on a Bruker ARX-300 spectrometer; two dimensional spectra were obtained using a standard Bruker software. EIMS spectra were obtained with direct inlet at 70 eV.

Filifolinyl senecionate

Fraction-3, was purified by CC (silica gel) using hexane-EtOAc (4:1) to afford 360 mg of (1) as a colourless oil; [a]_D²⁰-17.3 (CHCl₃, c 0.4); UV l_max (MeOH) nm (log e): 216 (4.22), 265 (4.13), 290 (3.15); FTIR n_max (KBr) cm^-1: 1750, 1720; HRMS m/z 386.209321 [M⁺] calculated for C₂₃H₃₀O₅ 386.209325; EIMS m/z (rel. Int.): 386 [M⁺] (10), 286 [M-C₅H₈O₂]⁺ (100); ¹H-NMR (300 MHz, CDCl₃): Table 1. ¹³C-NMR (100.6 MHz CDCl₃): Table 1.

Methanolysis of filifolinyl senecionate

Filifolinyl senecionate (50 mg) was dissolved in anhydrous acid methanol and kept at room temperature with stirring during 24 h. After work-up of the mixture, 30 mg of filifolinol were obtained and identified by direct comparison with an authentic sample ²⁾ (mmp, TLC, ¹H-NMR).

RESULTS AND DISCUSSION

The molecular formula C₂₃H₃₀O₅ for (1) was deduced from its exact mass [M⁺] at m/z 386.209321. The analysis of the NMR spectra (Table 1) gave a similar structure to that of filifolinol (2) ²⁾, but (1) is esterified with a five-carbon acid. Acid methanolysis of (1) yielded (2), confirming the spectroscopic results.

The structure of the acid moiety was determined through high-field two dimensional NMR techniques, in special, a long range 2D-INDEPT experiment (Table 2) showed that the acid moiety was senecioic acid.

In addition to the aromatic geranyl derivatives isolated from the resinous exudates of H. huascoense Johnston, H. stenophyllum H. & A. and H. filifolium Johnston ⁷⁾, we have conclusive evidence, obtained by HPLC coupled with UV-Vis spectroscopy ⁸⁾ that aromatic geranyl derivatives are also present in the resinous exudates of H. sinuatum (Miers) Johnston and H. chenopodiaceum var. chenopodiaceum (D.C.) Clos. The results of this communication are in agreement with the postulate that geranyl aromatic derivatives are accumulated in the glandular trichomes of Heliotropium spp. ⁷⁾.

The results of the antibacterial experiment are summarized in Table 3. Filifolinyl senecionate has shown antibacterial activity against Escherichia coli K-12. After irradiation of a solution of filifolinyl senecionate with UV at 366 nm, the antibacterial activity was enhanced by 100%. Harmane, a known phototoxic compound to bacteria and insects ⁶⁾, was used as standard, harmane after irradiation only shows 60% of enhancement of the antibacterial activity.

The phototoxic activity found in filifolinyl senecionate strengthens the fact that the exudates produced by glandular trichomes constitute the first line of resistance of plants against insect and microorganisms ⁹⁾.

ACKNOWLEDGEMENTS

This work was supported by FONDECYT (Chile) N 1990209 and by DICYT (Universidad de Santiago de Chile).

REFERENCES

1. I. Johnston, Contrib. Gray Herbar Harvard University, 81, 83 (1928).

2. R. Torres, L. Villarroel, A. Urzúa, G. Delle-Monache, E. Delle-Monache, E. Gacs-Baitz, Phytochemistry, 36, 250 (1994).

3. A. Urzúa, L. Villarroel, R. Torres, S. Teiller, Biochem. Syst. Ecol., 21, 744 (1993).

4. A. M. Janssen, J. J. C. Shaeffer, A. Baerheim-Svendsen, Planta Médica, 39, 395 (1987).

5. Organización Mundial de la Salud, (1989) Valoración Microbiológica de los Antibióticos, 3 Ed. Vol. 1, Ginebra pp. 155-162.

6. R. A. Larson, K. A. Marley, R. W. Tuverson, M. R. Berenbaum, Photochem. Photobiol., 48, 665 (1988).

7. A. Urzúa, B. Modak, L. Villarroel, R. Torres, L. Andrade, L. Mendoza, M. Wilkens, Bol. Soc. Chil. Quím., 45, 23 (2000).

8. A. Urzúa, B. Modak, L. Villarroel, R. Torres, L. Andrade, Biochem. Syst. Ecol., 26, 130 (1998).

9. L. M. Schooven, T. Jeremy, J. J. A. van Loon, (1998) Insect-Plant Biology from Physiology to Evolution, Chapman and Hall, London pp. 31-32.