New Hopane Triterpene from Eleocharis sellowiana ( Cyperaceae )

The genus Eleocharis R. Br. (Cyperaceae, Cyperoideae, Scirpeae) includes about 200 species, occurring in wet environments like swamps, lakes and rivers margins. Their aerial parts are formed by simple, not ramified stalks that end in a spiciform inflorescence formed by numerous very inconspicuous flowers. Their subterraneous parts are formed by roots and stem (called rhizome or stolon, depending on its form). E. sellowiana Kunth occurs in Mexico, Colombia, Paraguay and Brazil. This species is widely distributed in São Paulo State. As many other plants from wet environments like swamps, lakes and rivers margins, Eleocharis species are subject of only a few studies. According to the literature only E. dulcis Trin., E. coloradoensis (Britt.) Gilly, E. acuta R. Br., E. microcarpa Torr. and E. smallii L. had been subjected to phytochemical analysis before. In continuation of our phytochemical analysis of Eleocharis genus, the hexanic extracts of E. sellowiana (subterraneous and aerial parts) were subjected to a detailed chromatographic analysis resulting in the isolation of a new pentacyclic triterpene named 3α-hydroxy13α,17α,21β-hopan-15,19-dione (1) and E-phytyl hexadecanoate (2) (Figure 1). Results and Discussion


Introduction
The genus Eleocharis R. Br. (Cyperaceae, Cyperoideae, Scirpeae) includes about 200 species, occurring in wet environments like swamps, lakes and rivers margins.Their aerial parts are formed by simple, not ramified stalks that end in a spiciform inflorescence formed by numerous very inconspicuous flowers.Their subterraneous parts are formed by roots and stem (called rhizome or stolon, depending on its form).E. sellowiana Kunth occurs in Mexico, Colombia, Paraguay and Brazil.This species is widely distributed in São Paulo State. 1 As many other plants from wet environments like swamps, lakes and rivers margins, Eleocharis species are subject of only a few studies.According to the literature 2 only E. dulcis Trin., E. coloradoensis (Britt.)Gilly, E. acuta R. Br., E. microcarpa Torr.and E. smallii L. had been subjected to phytochemical analysis before.
In continuation of our phytochemical analysis of Eleocharis genus, 3 the hexanic extracts of E. sellowiana (subterraneous and aerial parts) were subjected to a detailed chromatographic analysis resulting in the isolation of a new pentacyclic triterpene named 3α-hydroxy-13α,17α,21β-hopan-15,19-dione (1) and E-phytyl hexadecanoate (2) (Figure 1).  1) suggested a hopane-like or a lupanelike triterpene skeleton.The broad singlet at δ H 3.44 (brs, H-3) together with the chemical shift of C-3 at δ C 75.9 (CH) in its 13 C NMR spectrum (Table 2) suggested the presence of   1).These correlations indicated that both carbonyl carbons should not be present at C-ring.Analyzing the other correlations obtained in the HMBC NMR experiment, it was possible to determine the presence of the carbonyls at C-15 (D-ring) and at C-19 (E-ring) (Figure 2).The relative configuration at C/D and at D/E rings fusions was deduced through the valuable information about correlations among the hydrogen spin systems at carbons C-16, C-20 and C-30 furnished by the 1D-TOCSY NMR experiment (Figure 3).Accordingly, when hydrogens H-20 were irradiated, it was possible to identify the hydrogens H-21 (q, J 6.0Hz), H-22 (oct, J 6.0 Hz), H-29 (s) and H-30 (s).On the other hand, when the hydrogens H-16 were irradiated, only the hydrogen H-17 was observed.The absence of polarization transference between H-17 and H-21 points out to a very small coupling constant between these hydrogens and consequently to a cis-D/E rings fusion with a β-isopropyl group.The C/D rings fusion should be a cis-fusion with the hydrogen H-13 in a α-position.Through the corresponding molecular model it is possible to see that the methyl group CH 3 -26 is under the C-19 carbonyl protection cone, which explains the lower chemical shift of CH 3 -26 (δ H 0.85) hydrogens when compared to those related for the 17α,21βhopane (δ H 0.95). 5In its NOESY-1D spectrum, it was observed that when CH 3 -27 (δ H 1.15) was irradiated increments were observed in H-7a (δ H 1.72, 1.78%), H-9a (δ H 1.90, 0.95%) and H-12a (δ H 1.52, 2.13%) but not in CH 3 -28 (δ H 0.94).On the other hand, when CH 3 -28 (δ H 0.94) was irradiated increments were just observed in H-22 (δ H 1.76, 1.66%), corroborating with a cis-C/D rings fusion.In its HR-EIMS spectrum, there are some important fragment ions 6 that corroborated with the hydroxyl group at C-3 (m/z 207, 16%, 1a and m/z 189, 100%, 1b) and with carbonyl groups location at D-and E-rings (m/z 318, 85%, 1c; m/z 300, 24%, 1d; m/z 303, 20%, 1e; m/z 277, 32%, 1f) (Figure 4).

Results and Discussion
Compound 2 (C 28 H 48 O), isolated from the hexanic extract of E. sellowiana (aerial part), was identified as Ephytyl hexadecanoate by comparison of their 1 H and 13 C NMR and MS data with those previously published. 7The occurrence of compound 2 in the genus Eleocharis is described here for the first time.

General
Thin layer chromatography (TLC): Silica gel 60 F 254 Al sheets (Merck); detection at 254 and 365 nm and with anisaldehyde in acidic ethanol solution, 8 CC = column chromatography. 1 H, 13 C NMR and 2D experiments Varian Inova-500 (Palo Alto, CA, USA) spectrometer at 11 tesla.Chemical shifts of the compounds were recorded in CDCl 3 solutions and were quoted relative to TMS for 1 H NMR (δ 0.0) and to CDCl 3 (δ 77.0) for 13 C NMR. Atributions: The triterpene numering (see Figure 1) is used in the results Table 1. 1 H NMR (500 MHz, CDCl 3 ) and observed correlation in HSQC (vicinal C-H) and in HMBC (long-range C-H) spectra (CDCl 3 , 11 Tesla) of 1 and discussion and for spectroscopic data.HREIMS experiment: VG Auto Spec 10000 Micromass (Manchester, UK) instrument with an ionizing potencial of 70 eV, m/z (rel.intensity in%), direct probe.

Plant material
Samples of E. sellowiana were collected in Campinas, Brazil, and identified by two of the authors (A.D.

Isolation
Part of the hexanic extract EsSH (300 mg) was subjected to flash CC over silica gel, 8 eluted first with CHCl 3 -Hex (4:1).The eluent polarity was increased by the gradual addition of chloroform and then methanol until reaching 100% of methanol, furnishing 159 fractions (15 After solvent evaporation, part of the hexanic extract EsAH (467.0 mg) was fractionated by successive preparative TLC run with CH 2 Cl 2 -MeOH-H 2 O (85:15:1).These furnished seven fractions, which were numbered according to their decreasing polarities.Part of the seventh fraction (174.1 mg) was fractionated by successive preparative TLC run with CH 2 Cl 2 -Hex (9:1).These eleven fractions were numbered according to their decreasing polarities.The fraction 7.11 was purified by preparative TLC continuously run with Hex-Et 2 O (39:1) during 100 min to afford 2 (4.0 mg) which was analyzed by its NMR and MS spectra data.
F. and M. C. E. A.). Voucher specimens (A.D. Faria et al. 1000 -E.sellowiana) have been deposited in the Herbarium of the Botany Department of the Biology Institute of Unicamp (UEC), Campinas-SP, Brazil.

Table 2 .
13C NMR data of 1 and 3 Figure 4. Principal fragments observed in MS spectra of 1.