Chemical components from the surface of

Biochemical Systematics and Ecology 35 (2007) 794e796 www.elsevier.com/locate/biochemsyseco

Chemical components from the surface of Haplopappus bustillosianus Alejandro Urzuá a,*, Breezy Iturra a, Betania Sebastian a, Melica Munõz b a

Laboratorio de Quı´mica Ecolo´gica, Facultad de Quı´mica y Biologıá, Universidad de Santiago de Chile, Casilla 40, Correo-33, Santiago, Chile b Museo Nacional de Historia Natural, Seccioń Botanica, Santiago, Chile Received 23 June 2006; accepted 19 March 2007

Keywords: Haplopappus bustillosianus; Asteraceae; Resinous exudate; Monoterpenes; Sesquiterpenes; Diterpenes; Flavonoids; Waxy coating; n-Alkanes

1. Subject and source Haplopappus is well represented in Chile with 63 or 85 species, depending on the author (Reiche and Philippi, 1902; Marticorena and Quezada, 1985). With few exceptions all species produce external resinous exudates on twigs and leaves (Marticorena and Quezada, 1985). The exudates are biosynthesised in specialised glands (trichomes) populating the surface of all aerial structures of the plants. In addition, other specialised structures produce the waxy coating and additional specialised secretory structures may also be involved. A representative sample of aerial parts of Haplopappus bustillosianus Remy (Asteraceae) was collected during the flowering season, November 2004, from a population found beside Lake Villarrica (IX Region, Chile 39 160 S, 72 000 W). Voucher specimen (SGO-152463) was deposited in the Herbarium National Natural History Museum, Santiago, Chile. 2. Previous work There are no previous published studies on the chemistry of H. bustillosianus. 3. Present study Chemical components from the surface of H. bustillosianus (1.5 kg) were obtained by dipping aerial parts in cold CH2Cl2 for 20e30 s. The extracts were concentrated to a solid residue (15 g, 1.0%). The surface plant extract (5.0 g) was fractionated by CC (silica gel) using pentane, pentaneeCH2Cl2 and CH2Cl2e MeOH to afford three fractions. * Corresponding author. Tel.: þ56 2 682 1048; fax: þ56 2 681 2108. E-mail address: [email protected] (A. Urzuá). 0305-1978/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2007.03.020

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The surface plant extract and the fraction containing compounds eluted with pentane (hydrocarbons) were analyzed in triplicate, by GCeMS using a FISONS MD-800, equipped with a HP Ultra-II capillary column (25 m 0.20 mm). The temperature of the injector was 285 C, and the temperature of the column was programmed to starting at 50 C for 2 min, followed by a rise to 280 C at 15 C min1. Then it was kept isothermally for 50 min. Helium was the carrier gas at 15 psi. Detection was done by EI. Compounds were identified by comparison of the retention time with standards and of the mass spectra with data from the NIST library (1998) linked to the mass detector. Only correlation indexes greater than 98% were accepted (Urzuá et al., 2000, 2004). Retention indexes were calculated to confirm identification when standards were not available. The following compounds were identified. Hydrocarbons: n-Alkanes (1.9%): C11H24; C12H26; C13H28; C14H30; C15H32; C16H34; C17H36; C18H38; C19H40; C20H42; C21H44; C22H46; C23H48; C24H50; C25H52; C26H54; C27H56; C28H58; C29H60; C30H62; C31H64; C32H66 and C33H68. In the hydrocarbons, C25H52; C27H56; C29H60 and C31H64 gave account of 58% of the fraction. Monoterpenes (trace amounts): a-pinene; b-pinene and a-linalool. Sesquiterpenes (trace amounts): copaene; isocaryophyllene; 3-cubebene; 4(15)-cubebene; humulatriene; a-bisabolol; 4,10(15)-cadinadiene; 4,9-cadinadiene and 1,10(4)-cadinadiene. Diterpenes (trace amounts): thunbergol. Miscellaneous compounds (trace amounts): p-hydroxyacetophenone. Extensive CC (silica gel) and preparative TLC from the fractions eluted with pentaneeCH2Cl2 and CH2Cl2eMeOH yield the following compounds: Flavonoids, 5,7-dihydroxy-3,6,40 -trimethoxyflavone (130 mg) and 5,7,40 -trihydroxy-3,6dimethoxyflavone (120 mg); diterpenoids, ent-3-cleroden-15-oic acid (populifolic acid) (230 mg) and ent-3-cleroden15-oic acid methyl ester (309 mg). Identification of the compounds was confirmed by HRMS, NMR (400 MHz) experiments (1H, 13C, DEPT, COSY, HSQC, HMBC, and NOESY) and by comparison with spectroscopic and physical data from the literature (Leitao et al., 1992; Lopez et al., 1987; Williams et al., 1994, 1999). 4. Chemotaxonomic and ecological significance More than 5% of the surface compounds of H. bustillosianus correspond to two flavonoids: 5,7-dihydroxy-3,6,40 trimethoxyflavone and 5,7,40 -trihydroxy-3,6-dimethoxyflavone. This is a large amount for the genus since only minute amounts of flavonoids have been identified from the surface of other species, such as Haplopappus deserticola, Haplopappus foliosus, Haplopappus rigidus, Haplopappus uncinatus and Haplopappus velutinus, and, whereas they have not been detected on the surface of Haplopappus diplopappus, Haplopappus illinitus, and Haplopappus schumannii (Morales et al., 2000; Urzuá et al., 1997; 2004; Urzuá, 2004). Flavonoids oxygenated in position 6, a substitution pattern not frequently found in this genus, have been reported in: Haplopappus bezanillanus, Haplopappus canescens, H. foliosus, Haplopappus rengifoanus, Haplopappus scrobiculatus (Bohm and Stuessy, 2001), H. uncinatus (Urzuá et al., 2004) and H. rigidus (Morales et al., 2000). In the case of H. foliosus, H. uncinatus and H. rigidus it was possible to show that 6-oxygenated flavonoids are leaf surface components. Although the composition profile of the hydrocarbon fraction of H. bustillosianus is similar to that of other species of Haplopappus, there is a difference in the yield of this fraction (Urzuá et al., 2004; Urzuá, 2004). The surface chemistry of species growing in the mountains contained from 25% to 50% hydrocarbons, whereas H. bustillosianus only yielded 1.9%. This value is lower than that of H. foliosus (8%) a species found growing in coastal area of the Pacific Ocean (IVeV Regions, Chile 32e34 S). Finally, an overview of the terpene composition of H. bustillosianus is in agreement with the results found for the surface chemistry of H. foliosus, H. uncinatus, H. velutinus, H. rigidus, H. deserticola, H. schumannii, H. illinitus and H. diplopappus: trace amounts of monoterpenes and sesquiterpenes and larger amounts of diterpenes, in this case, of the clerodane family which are common components of the genus together with labdane diterpenoids (Morales et al., 2000; Tojo et al., 1999; Urzuá et al., 1995, 2000, 2004; Urzuá, 2004). Even though some of these molecules and structural types are repeated among species, a clear terpene pattern implying chemosystematic correlations in the genus Haplopappus could not be found. Flavonoid and hydrocarbon fractions are of interest because of the well documented eco-physiological roles of these compounds. Exudate flavonoids protect plants from UV radiation, high temperature and hydric stress (Chaves et al., 1997). The wax coating of leaves protects plants from light, temperature, osmotic stress, physical damage, altitude and pollution (Shephered et al., 2006). Flavonoid biosynthesis is activated by plants as a response to exposition to UV radiation (Logemann et al., 2000), and their accumulation has been demonstrated to serve, among other roles, as a protective shield to leaves preventing

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molecular and cellular damage. For example, 5,4-dihydroxy-3,6,7,8-tetramethoxyflavone and 5,4-dihydroxy3,6,7,8,30 -pentamethoxyflavone, natural occurring compounds in Gnaphalium luteo-album, significantly increase their concentration in leaves after UV-B radiation exposition (Cuadra et al., 1997). A protective role is also attributed to the wax coating of leaves, which is partially composed by the n-alkanes fraction. This leaf covering structure not only serves as a filter for harmful radiation, but also avoids excessive loss of water of the plant by evapo-transpiration that would lead to stress. H. bustillosianus natural habitat is a lake area with an average annual precipitation of 1000 mm. These conditions contrast with those of other species of Haplopappus, especially those growing in the Andes, which are adapted to extremely dry conditions and present high proportions of n-alkanes in their surface chemistry. The extremely low yield of n-alkanes in H. bustillosianus is, then, consistent with its environmental conditions since this species is not susceptible to suffering water stress. An average ozone decline of around 2.5% per decade has been estimated for the band from 30 to 50 in the southern hemisphere (Harris et al., 2003), where Lake Viillarica is located (39 160 S). Also, recurrent episodes of ozonepoor air masses reaching mid-latitudes in South America have been documented (Lovengreen et al., 2000; Perez and Jaque, 1998). Because significant amounts of surface flavonoids are not characteristic of the genus Haplopappus, their presence in H. bustillosianus is consistent with the possibility of the species being repeatedly exposed to an excess of UV radiation. Acknowledgements This work was supported by Universidad de Santiago de Chile (DICYT) Chile. References Bohm, B.A., Stuessy, T.F., 2001. Flavonoids of the Sunflower Family (Asteraceae). Springer, New York. Cuadra, P., Harborne, J., Waterman, P.G., 1997. Phytochemistry 45, 1377. Chaves, N., Escudero, J.C., Gutierrez-Merino, C., 1997. J. Chem. Ecol. 23, 579. Harris, J.M., Oltmans, S.J., Bodeker, G.E., Stolarski, R., Evans, R.D., Quincy, D.M., 2003. Atmos. Environ. 37, 3167. Leitao, G.G., Kaplan, C.M.A., Galeffi, C., 1992. Phytochemistry 31, 3277. Logemann, E., Tavernaro, A., Schulz, W., Somssich, E.I., Hahlbrock, K., 2000. Proc. Natl. Acad. Sci. USA 97, 1903. Lopez, M.X.L., Bolzani, V., Da, S., Trevisan, M.V.L., 1987. Phytochemistry 26, 2781. Lovengreen, C., Fuenzalida, H., Villanueva, L., 2000. Atmos. Environ. 34, 4051. Marticorena, C., Quezada, M., 1985. Gayana 42, 33. Morales, G., Sierra, P., Borquez, J., Loyola, L.A., 2000. J. Chil. Chem. Soc. 49, 137. Perez, A., Jaque, F., 1998. Atmos. Environ. 32, 3665. Reiche, C., Philippi, F., 1902. Flora de Chile. vol. 3. Imprenta Cervantes, Santiago, Chile. Shephered, T., Wynne, G.D., 2006. New Phytol. 171, 469. Tojo, E., Rial, M.E., Urzuá, A., Mendoza, L., 1999. Phytochemistry 52, 1531. Urzuá, A., Tojo, E., Soto, J., 1995. Phytochemistry 38, 555. Urzuá, A., Mendoza, L., Andrade, L., Miranda, B., 1997. Biochem. Syst. Ecol. 25, 683. Urzuá, A., Andrade, L., Jara, F., 2000. Biochem. Syst. Ecol. 28, 491. Urzuá, A., Contreras, R., Jara, P., Avila, F., Suazo, M., 2004. Biochem. Syst. Ecol. 32, 215. Urzuá, A., 2004. J. Chil. Chem. Soc. 49, 137. Williams, A.C., Hoult, J.R.S., Harborne, B.J., Greenham, J., Eagles, J., 1994. Phytochemistry 38, 267. Williams, A.C., Harborne, B.J., Hoult, J.R.S., Geiger, H., 1999. Phytochemistry 51, 471.