New hopane triterpene from Eleocharis sellowiana (Cyperaceae)

Abstracts

A new triterpene named 3alpha-hydroxy-13alpha,17alpha,21beta-hopan-15,19-dione and E-phytyl hexadecanoate were isolated from the hexanic extract of Eleocharis sellowiana. NMR and MS experiments determined the molecular structures.

Eleocharis; Cyperaceae; hopane-like triterpene

Do extrato hexânico de Eleocharis sellowiana foram isolados o novo triterpeno 3alfa-hidroxi-13alfa,17alfa,21beta-hopano-15,19-diona e hexadecanoato de E-fitila, caracterizados por dados de RMN e EM.

SHORT REPORT

New hopane triterpene from Eleocharis sellowiana (Cyperaceae)

Ana Lúcia T. G. RuizI, II, ^*; Aderbal F. Magalhães^I; Aparecida D. Faria^III; Eva G. Magalhães^I; Maria do Carmo E. Amaral^III

^IInstituto de Química, Universidade Estadual de Campinas, CP 6154, 13084-862 Campinas - SP, Brazil

^IICentro Pluridisciplinar de Pesquisas Químicas, Biológicas e Agrárias, Universidade Estadual de Campinas, CP 6171, 13083-970 Campinas - SP, Brazil

^IIIInstituto de Biologia, Universidade Estadual de Campinas, CP 6109, 13083-970 Campinas - SP, Brazil

ABSTRACT

A new triterpene named 3a-hydroxy-13a,17a,21b-hopan-15,19-dione and E-phytyl hexadecanoate were isolated from the hexanic extract of Eleocharis sellowiana. NMR and MS experiments determined the molecular structures.

Keywords:Eleocharis, Cyperaceae, hopane-like triterpene

RESUMO

Do extrato hexânico de Eleocharis sellowiana foram isolados o novo triterpeno 3a-hidroxi-13a,17a,21b-hopano-15,19-diona e hexadecanoato de E-fitila, caracterizados por dados de RMN e EM.

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 literature² 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 3a-hydroxy-13a,17a,21b-hopan-15,19-dione (1) and E-phytyl hexadecanoate (2) (Figure 1).

Results and Discussion

Compound 1 was deduced as having an elemental formula of C₃₀H₄₈O₃ by HREI-MS (observed M^+•= 456.3607; required M^+•= 456.3604). The presence of five signals as singlets (d_H 0.85, d_H 0.89, d_H 0.94, d_H 0.97 and d_H 1.15) and two signals as doublets (d_H 0.86, d, J 7.0Hz, CH₃-29, and d_H 1.00, d, J 7.0Hz, CH₃-30) attributed to eight methyl groups in its ¹H NMR spectrum (Table 1) suggested a hopane-like or a lupane-like triterpene skeleton. The broad singlet at d_H 3.44 (brs, H-3) together with the chemical shift of C-3 at d_C 75.9 (CH) in its ¹³C NMR spectrum (Table 2) suggested the presence of a 3a-OH group⁴ (Table 2) while the signals in at d_C 215.7 (C-15) and d_C 224.1 (C-19) showed correlation with the methyl hydrogens at CH₃-27 e CH₃-28 in the HMBC NMR experiment (Table 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 b-isopropyl group. The C/D rings fusion should be a cis-fusion with the hydrogen H-13 in a a-position. Through the corresponding molecular model it is possible to see that the methyl group CH₃-26 is under the C-19 carbonyl protection cone, which explains the lower chemical shift of CH₃-26 (d_H 0.85) hydrogens when compared to those related for the 17a,21b-hopane (d_H 0.95).⁵ In its NOESY-1D spectrum, it was observed that when CH₃-27 (d_H 1.15) was irradiated increments were observed in H-7a (d_H 1.72, 1.78%), H-9a (d_H 1.90, 0.95%) and H-12a (d_H 1.52, 2.13%) but not in CH₃-28 (d_H 0.94). On the other hand, when CH₃-28 (d_H 0.94) was irradiated increments were just observed in H-22 (d_H 1.76, 1.66%), corroborating with a cis-C/D rings fusion. In its HR-EIMS spectrum, there are some important fragment ions⁶ 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).

Compound 2 (C₂₈H₄₈O), isolated from the hexanic extract of E. sellowiana (aerial part), was identified as E-phytyl hexadecanoate by comparison of their ¹H and ¹³C NMR and MS data with those previously published.⁷ The occurrence of compound 2 in the genus Eleocharis is described here for the first time.

Experimental

General

Thin layer chromatography (TLC): Silica gel 60 F₂₅₄ Al sheets (Merck); detection at 254 and 365 nm and with anisaldehyde in acidic ethanol solution,⁸ CC = column chromatography. ¹H, ¹³C NMR and 2D experiments Varian Inova-500 (Palo Alto, CA, USA) spectrometer at 11 tesla. Chemical shifts of the compounds were recorded in CDCl₃ solutions and were quoted relative to TMS for ¹H NMR (d 0.0) and to CDCl₃ (d 77.0) for ¹³C NMR. Atributions: The triterpene numering (see Figure 1) is used in the results 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. 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.

Extraction

The collected plant (Es = E. sellowiana) was separated into aerial (270.0g, EsA) and subterraneous (428.0g, EsS) parts. Extracts of fresh subterraneous part were obtained by maceration with ethanol. The resultant extracts were combined, concentrated and diluted with H₂O to get an aqueous EtOH solution, which was partitioned with hexane (EsSH, 358.4 mg) and CHCl₃ (EsSC, 1303.4 mg); the respective aq. EtOH layers were then named EsSHE (3010.0 mg). The air-dried milled aerial part (named EsA, 95.8g) was successively extracted in a Soxhlet apparatus with hexane (EsAH, 2249.3 mg), CH₂Cl₂ (EsAD, 447.9 mg) and MeOH (EsAM, 3052.0 mg).

Isolation

Part of the hexanic extract EsSH (300 mg) was subjected to flash CC over silica gel,⁸ eluted first with CHCl₃-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 mL), which were reduced to 21 groups after TLC. Group 3 (29.4 mg, fractions 7 and 8), after successive preparative TLC run with CHCl₃-MeOH (40:1) and with Et₂O-Hex (2:1), afforded 1 (4.0 mg).

3a-hydroxy-13a,17a,21 b-hopan-15,19-dione (1)

It was obtained as a colorless oil; ¹H NMR (CDCl₃, 500MHz): Table 2; ¹³C NMR (CDCl₃, 125MHz): Table 3; HREI-MS (70eV), m/z (%): 456.3607 (2), 441.3680 (9), 318.2508 (85), 303.2231 (20), 300.2370 (24), 277.2109 (32), 233.1865 (12), 207.1671 (16), 189.1581 (100), 177.1566 (7), 175.1408 (38), 152.1162 (10), 139.1056 (17), 137.1275 (9), 135.1118 (62), 121.0952 (52), 109.0966 (41), 107.0806 (56), 97.0590 (84), 95.0801 (63), 93.0646 (48).

After solvent evaporation, part of the hexanic extract EsAH (467.0 mg) was fractionated by successive preparative TLC run with CH₂Cl₂-MeOH-H₂O (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 CH₂Cl₂-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-Et₂O (39:1) during 100 min to afford 2 (4.0 mg) which was analyzed by its NMR and MS spectra data.

Acknowledgments

The authors are grateful to FAPESP for financial support and for the scholarship awarded to Ana Lúcia T.G. Ruiz. Dr. Maria do Carmo E. do Amaral also thanks CNPq for a research grant.

Supplementary Information

Supplementary data are available free of charge at http://jbcs.sbq.org.br, as PDF file.

REFERENCES

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Received: September 17, 2004

Published on the web: April 28, 2006

FAPESP helped in meeting the publication costs of this article.

Supplementary Information

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Publication Dates

• Publication in this collection
  25 Aug 2006
• Date of issue
  Aug 2006

History

• Received
  17 Sept 2004