Revista Brasileira de Farmacognosia Brazilian Journal of Pharmacognosy 22(3): 482-489, May/Jun. 2012 Chemical composition and histochemistry of Sphagneticola trilobata essential oil Cleber José da Silva,1,2 Luiz C. A. Barbosa,*,1 Antonio J. Demuner,1 Ricardo M. Montanari,1 Dayana Francino,2 Renata M. S. A. Meira,2 Ana Olívia de Souza3 Article 1Departamento de Química, Universidade Federal de Viçosa, Brazil, 2Departamento de Biologia Vegetal, Universidade Federal de Viçosa, Brazil, 3Laboratório de Bioquímica e Biofísica, Instituto Butantan, Brazil. Received 29 Jun 2011 Accepted 14 Sep 2011 Abstract: Anatomical and histochemical investigations of Sphagneticola trilobata (L.) Available online 17 Jan 2012 Pruski, Asteraceae, secretory structures in leaves and stems and the seasonal variation of essential oils were carried out. Histochemical techniques enabled the specifi c Keywords: location of the essential oil accumulation in the internal (canals) and external structures essential oils (trichomes). Histochemical analysis showed that the secretory trichomes produced seasonal variation steroids. The highest yield was obtained from plants collected in winter, when it was secretory structures registered low temperature and precipitation. The essential oil was characterized by Sphagneticola trilobata high percentage of hydrocarbon sesquiterpenes, hydrocarbon monoterpenes and low levels of oxygenated sesquiterpenes. The major components were germacrene D (11.9- 35.8%), α-phellandrene (1.4-28.5%), α-pinene (7.3-23.8%), E-caryophyllene (4.6- 19.0%), bicyclogermacrene (6.0-17.0%), limonene (1.8-15.1%) and α-humulene (4.0- ISSN 0102-695X http://dx.doi.org/10.1590/S0102- 11.6%). The percentage of most of the individual constituents present in S. trilobata 695X2012005000012 essential oil changed signifi cantly during the months. Introduction in specialized secretory structures such as ducts and glandular trichomes (Fahn, 1979). The biosynthesis of Sphagneticola trilobata (L.) Pruski, Asteraceae, these metabolites can be infl uenced by external factors Heliantheae, is the accepted name of a Brazilian native like temperature, season, number of hours of sunlight, herb that naturally grows in coastal regions, barren lands and soil composition (Martins et al., 1997; Freire et and forests, or as weed in crops, in many countries. This al., 2006; Demuner et al., 2011). In many cases genetic species has several synonyms, and the most common in variability can also result in plant of different chemotypes Brazil is Wedelia paludosa DC. In Australia it is known as (Castro et al., 2004; Silva et al., 2007; Lima et al., 2008). Singapore daisy and in some other countries it is known as Therefore, the seasonal variation and anatomical study of Wedelia, trailing or creeping daisy, water zinnia and rabbit’s S. trilobata is an important approach for the optimization paw (Meena et al., 2011). In folk medicine, S. trilobata is of crop, post-harvest and extraction techniques, toward the employed to treat backache, muscle cramps, rheumatism, potential commercial application of this natural resource. stubborn wounds, sores and swellings, and arthritic painful In addition, the chemical and anatomical knowledge about joints (Arvigo & Balik, 1993). Anticonceptive activity was this species can be useful for taxonomic resolution of this described for some extracts and the isolated compounds, controversial plant group. kaurenoic acid and luteolin, from S. trilobata (Block et Many plants of the Heliantheae tribe are al., 1998). Trypanosomicidal activity was observed for recognized as weeds, such as Bidens pilosa (Ballard, the ethanol extract (Chiari et al. 1996) and the bioassay- 1986), Parthenium hysterophorus (Kohli et al., 2006) and directed fractionation leads to isolation of the diterpenes also S. trilobata. The well-documented phytotoxic activity kaurenoic and grandifl orenic acids (Batista et al. 1999). of the essential oils can be related to the invasive ability The antimicrobial activity was demonstrated for the of these plants and others (Vokou et al., 2003; Barbosa et hexane and ethyl acetate extracts of the aerial parts against al., 2007). Thus, the study of this activity in S. trilobata Gram-positive and Gram-negative bacteria, but not against essential oil is important for understanding its ability to yeasts and fungi (Taddei & Rosas-Romero, 1999). adapt to different habitats and its invasive behavior in In many Asteraceae species, the secondary crops. metabolites that compose the essential oils are synthesized In this context, in the present investigation 482 Chemical composition and histochemistry of Sphagneticola trilobata essential oil Cleber José da Silva et al. we describe the first study of the chemical composition the chromatograms. of essential oils from stem and leaves of S. trilobata, and report on the histochemical localization of the oils Gas chromatography-Mass spectrometry (GC-MS) producing glands. The GC-MS unit (model GCMS-QP5050A, from Material and Methods Shimadzu, Japan) was equipped with a DB-5 fused silica column (30 m x 0.22 mm i.d., film thickness 0.25 μm) and Plant material and meteorological data interfaced with an ion trap detector. Oven and injector temperatures were as described above; transfer line Leaves of Sphagneticola trilobata (L.) Pruski, temperature, 240 ºC; ion trap temperature, 220 ºC; carrier Asteraceae, were collected every month, from September gas, He at a flow rate of 1.8 mL min-1; injector temperature 2008 to October 2009 always between 8 and 9 a.m. The 220 °C, detector temperature 240 °C; column temperature specimens were grown in the arboretum of the Herbarium, was programmed to start at 55 °C (isothermal for 2 min), at the Federal University of Viçosa (UFV), Minas Gerais with an increase of 3 °C min-1, to 240 °C, isothermal at state, Brazil. The materials were identified, herborized 240 °C for 15 min; injection of 1.0 μL (1% w/v in CH2Cl2); and a voucher specimen has been deposited in the VIC split ratio 1:10; column pressure of 100 kPa; ionization Herbarium (registration number is 32.484) of the Plant energy, 70 eV; scan range, 29-450 u; scan time, 1 s. The Biology Department, UFV. The registered values of identity of each component was assigned by comparison temperature and precipitation used in this study have of their retention indexes (RI), relative to a standard been provided by the National Institute of Meteorology of alkane series (C9-C27) and also by comparison of its mass Brazil. spectrum with either reference data from the equipment database (Wiley 7) or from the literature (Adams, 2007). Essential oil extraction Light microscopy and histochemical analysis Leaves were collected separately, in a completely randomized way, from the population under investigation. Small fresh sections of leaves and stems were fixed Each sample was subdivided into three portions of 100 in formalin-acetic acid-ethanol 50% (FAA) (Johansen, g each, chopped and then subjected to a three hours 1940). The material was then dehydrated, embedded in hydrodistillation in a Clevenger-type apparatus. The paraffin, sliced transversely and longitudinally with a rotary resulting oils were weighed and the reported yields were microtome (4-5μm) model RM2155 (Leica, Microsystems calculated with respect to dry matter mass. All distillations Inc., Deerfield, USA), and stained with safranin and astra were repeated three times and the oils produced in blue. Preparations were mounted in Permount (Fisher these processes were stored under nitrogen atmosphere, Scientific Co., Pittsburgh, PA). maintained at -4 oC, until they were analyzed by gas Fresh mature leaves were sectioned transversely chromatography coupled to a mass spectrometry (GC- and longitudinally using a manual operated table microtome MS). Leaf dry matter mass was calculated by drying (LPC, Rolemberg and Bhering Comércio e Importação each sample (2 g, held at 103±2 ºC until constant mass) LTDA, Belo Horizonte, Brazil). according to published methods (ASAE, 2000). Each The main classes of metabolites in the secreted determination was carried out in triplicate. material were investigated in fresh sections, using the following histochemical tests: Sudan Red (Johansen, Gas chromatography 1940), Nile Blue (Jensen, 1962) for neutral and acidic lipids and Nadi reagent (David & Carde, 1964) for terpenoids. GC analyses were carried out with a GC-17A For all tests standard control procedures were carried out Series instrument (Shimadzu, Japan) equipped with a flame simultaneously using the same procedures. ionization detector (FID). Chromatographic conditions Optical analysis and photographic documentation were as follows: DB-5 fused silica capillary column (30 were accomplished using a microscope (Olympus AX70) m x 0.22 mm, 0.25 μm film thickness); carrier gas, 2N at equipped with a U-Photo photographic system and digital a flow rate of 1.8 mL min-1; injector temperature 220 °C, camera (Diagnostic Instruments Work, model Camera detector temperature 240 °C; column temperature was Spot Insight). programmed to start at 55 °C (isothermal for 2 min), with For the fluorescence microscopy investigation, an increase of 3 °C min-1, to 240 °C, isothermal at 240 °C fresh material was treated with antimony trichloride for for 15 min; injection of 1.0 μL (1% w/v in CH2Cl2); split steroids and neutral red for total lipids (Jensen 1962). The ratio 1:10; column pressure of 115 kPa. The analyses were epifluorescence microscope (Olympus BX60) equipped carried out in triplicate and the amount of each compound with a UV filter (WU: 330-385 nm), a dichroic mirror (400 was expressed as a relative percentage of the total area of nm) and a barrier filter (420 nm) were used. Rev. Bras. Farmacogn. Braz. J. Pharmacogn. 22(3): May/Jun. 2012 483 Chemical composition and histochemistry of Sphagneticola trilobata essential oil Cleber José da Silva et al. Results and discussion germacrene D (11.9-35.8%), α-phellandrene (1.4-28.5%), α-pinene (7.3-23.8%), E-caryophyllene (4.6-19.0%), Volatile oil yields bicyclogermacrene (6.0-17.0%), limonene (1.8-15.1%) and α-humulene (4.0-11.6%).