MACNBR   00242
MUSEO ARGENTINO DE CIENCIAS NATURALES "BERNARDINO RIVADAVIA"
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
Unmasking the scent of Hypsiboas pulchellus (Anura: Hylidae) using in vivo headspace solid phase micro extraction
Autor/es:
BRUNETTI, ANDRÉS E.; CABRERA, GABRIELA M.; FAIVOVICH, JULIÁN; MERIB, JOSIAS; CARASEK, EDUARDO; BARBARÁ, JANAINA; COLOMBO, PATRICK; CARAMÃO, ELINA; ZINI, CLAUDIA
Lugar:
João Pessoa
Reunión:
Simposio; ExTech® 2013 15th International Symposium on Advances in Extraction Technologies; 2013
Resumen:
Introduction The skin of amphibians is a very dynamic organ that has diverse functions like maintenance of homeostasis, chemical defense against predators and microorganisms, and possibly chemical communication. It has been studied for decades by different groups all over the world, due to the high diversity of compounds that are secreted by the glands located in the dermis (1). Five types of molecules have been reported for amphibian skin: amines, peptides, alkaloids, bufodienolides, and tetrodotoxines (2). Volatile components are another type of molecules emitted by some species of amphibians, which are promptly perceived by human beings because of their characteristic and diverse odors. However, only three scientific articles have succeeded in the detection of volatile compounds emitted by amphibians; and two of them have chosen solid phase micro extraction (SPME) technique as an effective extraction tool (3?5). Hypsiboas pulchellus is a common species of frog with a wide geographical distribution, and it is well known by herpetologist because it releases a strong odor when handled in its natural environment (6). The aim of present work is to detect and tentatively identify the volatile molecules secreted by H. pulchellus, using headspace (HS) SPME, and evaluate the possible role of these volatile compounds, according to their molecular structure. Experimental Frogs were collected at the state of RS, Brazil. They were housed in acrylic aquarium, provided with clean water, food, and 12:12 h darklight period. Their volatiles were sampled by placing two individuals in a sealed 30 mL clear glass vial device. The bottom part of the device, where the frogs were located, was placed in a water bath, and its top, where the fiber was placed, was cooled. A gentle electrical stimulation was applied to stimulate the release of secretions. Some individuals were sacrificed using approved procedures and their skins were removed, extracted and analyzed through HSSPME and a Shimadzu GC-MS QP-2010 Plus gas chromatography with mass spectrometric detection (GC/MS). Different extraction conditions were necessary to achieve the necessary sensitivity for the detection of some of the compounds present in the frogs´ secretions (different fiber coatings, extraction times, and live frogs vs. skins alone). The chromatographic column employed was a Restek Rtx-5MS column (30m x 0.25mm x 0.25μm). Compounds were tentatively identified comparing experimentally determined mass spectra and linear temperature programmed retention index (LTPRI) with literature data. Results Nine low molecular weight compounds were tentatively identified: two alkenes, one aldehyde, two ketones, one alcohol, one ester, and two sulfides. Several chromatographic were performed in the scan mode generating total ion current chromatograms (TIC) and a preliminary assessment of tentatively identified compounds was performed. The most relevant ions of the tentatively identified compounds were employed to obtain selected ion chromatograms (SIC) in order to eliminate interferents and also to obtain an enhanced signal. Positive identification of the volatile compounds emitted by frogs will be accomplished with commercially available compounds in the near future. It is expected that the positive identification of frogs´ volatiles will allow the search for a relation between molecular structures and the well known odor of these amphibians. Fig 2. Selected ion current chromatograms (SIC) from most relevant ions of the compounds tentatively identified by TIC using PDMS/DVB/Carboxen fiber and GC/MS. (A) after 5 min of extraction. (B) After 40 min of extraction. Tentative identification of compounds is the following: 1 and 2-alkenes, 3-aldehyde, 4 and 5-ketones, 7 and 9- sulfides. Compounds 6 and 8 correspond to alcohol and ester respectively (not shown). Lower intensity traces (gray line) in both chromatograms correspond to a blank experiment, where frogs were not stimulated. Sensitivity for compounds 1 and 2 was enhanced after 5 min extraction (A). A higher sensitivity was achieved for compounds 3 to 5, 7 and 9 (Fig 2B compared to Fig. 2A). Compounds 6 and 8 were only observed when the headspace of skins was extracted (chromatogram not shown). Present findings constitute an important contribution to the knowledge of the volatile components emitted by frogs. Moreover, their tentative identification is an exciting starting point to study their biological function for this species in particular and as potential medicines. Also it will allow evaluating if these molecules are synthesized or if they come from dietary uptake. References 1. Toledo R. C., & Jared C. 1995. Comp. Biochem. Physiol. 111:1?29. 2. Erspamer V. 1994. In H. Heatwole and G.T.Barthalmus (eds), Amphibian biology, vol. 1: 178?350. Surrey: Beatty & Sons. 3. Smith B. P. C., Tyler M. J., Williams B. D., & Hayasaka Y. 2003. J. Chem. Ecol. 29:2085? 2100. 4. Poth, D., Wollenberg, K.C., Vences, M., Schulz, S. 2012. Angew. Chem. Int. Ed. 51: 2187?2190. 5. Smith, B.P., Zini, C.A., Pawliszyn, J., Tyler, M.J., Hayasaka, Y., Williams, B., Caramao, E.B. (2000). Chem. Ecol. 17: 215-225. 6. Gallardo J. M. 1961. Ciencia e Investigación XVII (3):63?69.