Biochemical Systematics and Ecology 34 (2006) 757e759 www.elsevier.com/locate/biochemsyseco

Melampolides from Ichthyothere terminalis (Asteraceae, Heliantheae) Ricardo Stefani a,b, Fernando Batista Da Costa b,* a

Departamento de Quı´mica, Faculdade de Filosofia, Cieˆncias e Letras de Ribeir~ao Preto, Universidade de S~ao Paulo (USP), Av. Bandeirantes 3900, 14040-901 Ribeir~ao Preto, SP, Brazil b Laborato´rio de Farmacognosia, Faculdade de Cieˆncias Farmaceˆuticas de Ribeir~ao Preto, USP, Av. do Cafe´ s/n, 14040-903 Ribeir~ao Preto, SP, Brazil Received 29 November 2005; accepted 27 May 2006

Keywords: Ichthyothere terminalis; Melampodiinae; Heliantheae; Asteraceae; Melampolides; Sesquiterpene lactones

1. Subject and source Ichthyothere terminalis (Spreng.), tribe Heliantheae, was collected by F.B. Da Costa on February 2002 at Fazenda
Agua Limpa, DF, Brazil, and identified by Prof. E.E. Schilling (University of Tennessee, TN, USA). A voucher specimen (collector number FBC #80) is deposited at the herbarium of Departamento de Biologia, Faculdade de Filosofia, Cieˆncias e Letras de Ribeir~ao Preto, Ribeir~ao Preto, SP, Brazil, under the code SPRF 4442. 2. Previous work Individuals of I. terminalis (voucher RMK 8630) have been previously investigated (Bohlmann et al., 1982), but no mention was made about the geographic origin of the species. In that study, the roots afforded kaurene derivatives, while the aerial parts afforded several sesquiterpene lactones (STLs) of the melampolide type. This subgroup of STLs is representative of this genus, since an earlier investigation of Ichthyothere ulei (Bohlmann et al., 1982) afforded the same type of compounds, but again there was no mention of its geographic origin. The occurrence of other secondary metabolite classes in I. terminalis, such as polyacetylenes (ichthyothereol and its acetate, Cascon et al., 1965) and flavonol derivatives (Bohm and Stuessy, 1982) has also been reported. 3. Present work A leaf rinse extract of the entire air dried leaves (32.8 g) of I. terminalis was prepared with CH2Cl2 at room temperature for 30 s. After solvent evaporation under reduced pressure, the extract was resuspended in a mixture of MeOH:H2O (7:3) to yield a residue (805 mg), which was chromatographed in a glass column over silica gel (60H, * Corresponding author. Tel.: þ55 16 3602 4312; fax: þ55 16 3602 4879. E-mail address: [email protected] (F.B. Da Costa). 0305-1978/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2006.05.009

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R. Stefani, F.B. Da Costa / Biochemical Systematics and Ecology 34 (2006) 757e759

Merck, Art. 7736, 100e200 mesh ASTM) and eluted with n-hexane with increasing amounts of EtOAc. Fractions (250 mL) were collected as follows: 1 (n-hexane, 4 mg), 2 (n-hexane:EtOAc 9:1, 102 mg), 3 (n-hexane:EtOAc 7:3, 88 mg), 4 (n-hexane:EtOAc 1:1, 127 mg), 5 (n-hexane:EtOAc 3:7, 62 mg), and 6 (EtOAc, 28 mg). All the fractions were monitored by IR spectral analysis and fraction 4 showed strong absorption at 1750 cm
1 indicating the presence of a carbonyl group of a g-lactone. It was purified using HPLC (Shimadzu LC 10Avp, UVDAD detector SPD-M10Avp, and a Shimadzu column C18, 5 mm, 46  250 mm) using a mixture of MeOH:H2O 48:52 as mobile phase and a flow rate of 1.0 mL/min. A total of 10 fractions were collected and submitted to 1H NMR analysis on a Bruker ARX 400 spectrometer (400 MHz for 1H, CDCl3). Fractions 4.3 (1, 8 mg) and 4.7 (2, 10 mg) consisted of pure STLs. Structure identification was made by direct comparison of 1H NMR data with data from the literature and the present data are in good agreement with those reported for both 1 (Inoue et al., 1995; Krishnaswamy and Ramji, 1995) and 2 (Bohlmann et al., 1982). The sesquiterpene lactone 1 was identified as the melampolide enhydrin, which so far has not been isolated from this genus, while sesquiterpene lactone 2 was previously isolated from I. terminalis (Bohlmann et al., 1982) and identified as 2a-hydroxylongipilin acetate. An HPLC UV-DAD run was carried out in the same conditions described above, and showed that the melampolide enhydrin is the major compound of the leaf rinse extract. As the current 1 H NMR data set of enhydrin (1) is more comprehensive than that previously reported, they are listed below. Enhydrin (1). 1H NMR (400 MHz, CDCl3): d 7.15 (1H, dd, J ¼ 6.3, 9.7 Hz; H-1), d 6.72 (1H, dd, J ¼ 1.8, 8.5 Hz; H-8), d 6.34 (1H, d, J ¼ 3.5 Hz; H-13a), d 5.86 (1H, d, J ¼ 8.5 Hz; H-9), d 5.84 (1H, d, J ¼ 3.5 Hz; H-13b), d 4.27 (1H, d, J ¼ 9.5 Hz; H-6), d 3.80 (3H, s; OMe), d 3.02 (1H, d, J ¼ 5.0 Hz; H-30 ), d 2.67 (1H, d, J ¼ 9.5 Hz; H-5), d 2.98 (1H, dddd, J ¼ 1.8, 3.5, 3.5, 9.5 Hz; H-7), d 2.45 (1H, m; H-2a), d 2.34 (1H, m; H-2b), d 2.05 (3H, s; OAc), d 2.00 (1H, m; H-3a), d 1.98 (1H, m; H-3b), d 1.72 (3H, s; H-15), d 1.35 (3H, s; H-50 ), d 1.18 (3H, d, J ¼ 5.0 Hz; H-40 ). 14 COOMe OAc R2

1

10

OR1

9 8

2 5

3

7 6

4

13 11

O O

15

12

O

1

2

4'

4' 3'

3' O

R1

2'

2'

1'

1' 5'

5'

O R2

H

O OH

4. Chemotaxonomic significance The genus Ichthyothere is represented by 25 species distributed in South and Central America (Rauscher, 2002) and its taxonomic classification is still discussed. This genus was first placed by Hoffmann in the subtribe Melampodiinae (Hoffmann, 1890) and then moved by Stuessy to the subtribe Milleriinae (Stuessy, 1977). Robinson, based on morphological characters of the genus, placed it again in the subtribe Melampodiinae (Robinson, 1981), while Karis and

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Ryding (1994) in their morphological cladistic analysis of the Heliantheae, classified Ichthyothere as being of uncertain subtribal classification. A recent study involving molecular phylogenetics (Rauscher, 2002) suggests that Ichthyothere is a genus with close relationships to the Espeletia group. Although the exact relationship is still not known, it is clear that the Espeletia group is a member of Melampodiinae. Moreover, there is strong evidence that Ichthyothere should remain in Melampodiinae. It is quite evident that a divergence in opinion about the classification of the genus Ichthyothere does exist and thus the STL chemistry of Ichthyothere can be helpful for its classification. So far, only two species of Ichthyothere have been chemically investigated, and the results support the placement of Ichthyothere in the subtribe Melampodiinae, since the same type of STLs (melampolides) found in Ichthyothere are widespread in this subtribe. This includes STLs common to other genera of Melampodiinae, such as enhydrin, which was previously reported in Enhydra, Smallanthus and Polymnia (Krishnaswamy and Ramji, 1995; Inoue et al., 1995; Lin et al., 2003). In our study, these two melampolides were identified in a leaf rinse extract of a Brazilian specimen, followed by HPLC UV-DAD analysis. This technique has been proved to be useful for the investigation of the STL chemistry of the Heliantheae (Da Costa et al., 2001; Schorr et al., 2002). In addition, the melampolide enhydrin is an important compound from medicinal plants and shows biological activities (Feltenstein et al., 2004; Siedle et al., 2004). Acknowledgements The authors are grateful to CAPES, CNPq and FAPESP for financial support and E.E. Schilling for plant identification. References Bohm, B., Stuessy, T.F., 1982. Phytochemistry 21, 2761. Bohlmann, F., Jakupovic, J., Schuster, A., King, R.M., Robinson, H., 1982. Phytochemistry 21, 2317. Cascon, S.C., Mors, W.B., Tursch, B.M., Aplin, R.T., Durham, L.J., 1965. J. Am. Chem. Soc. 87, 5237. Da Costa, F.B., Schorr, K., Arakawa, N.S., Schilling, E.E., Spring, O., 2001. J. Braz. Chem. Soc. 12, 403. Feltenstein, M.W., Schu¨hly, W., Warnick, J.E., Fischer, N.H., Sufka, K.J., 2004. Pharmacol. Biochem. Behav. 79, 299. Hoffmann, O., 1890. Compositae. In: Engler, A., Prantl, K. (Eds.), Die Natu¨rlichen Pflanzenfamilien, vol. 4. Engelmann, Leipzig, pp. 210e267. Inoue, A., Tamogami, S., Kato, H., Nakazato, Y., Akiyama, M., Kodama, O., Akatsuka, T., Hashidoko, Y., 1995. Phytochemistry 39, 845. Karis, P.O., Ryding, O., 1994. Tribe Heliantheae. In: Bremer, K. (Ed.), Asteraceae: Cladistics and Classification. Timber Press, Portland, pp. 559e 624. Krishnaswamy, N.R., Ramji, N., 1995. Phytochemistry 38, 433. Lin, F., Hasegawa, M., Kodama, O., 2003. Biosci. Biotechnol. Biochem. 67, 2154. Rauscher, J.T., 2002. Am. J. Bot. 89, 1074. Robinson, H., 1981. Smithson. Contrib. Bot. 51, 1. Schorr, K., Garcia-Pin˜eres, A.J., Siedle, B., Merfort, I., Da Costa, F.B., 2002. Phytochemistry 60, 733. Siedle, B., Garcia-Pin˜eres, A.J., Murillo, R., Schulte-Mo¨nting, J., Castro, V., Ru¨ngeler, P., Klaas, C.A., Da Costa, F.B., Kisiel, W., Merfort, I., 2004. J. Med. Chem. 24, 6042. Stuessy, T.F., 1977. Heliantheae e systematic review. In: Heywood, V.H., Harborne, J.B., Turner, B.L. (Eds.), The Biology and Chemistry of the Compositae. Academic Press, New York, pp. 621e671.