Triterpenes from the flowers of Gochnatia polymorpha subsp. floccosa

Abstract

Phytochemical study of the flowers of Gochnatia polymorpha subsp. floccosa, Asteraceae, yielded eleven known triterpenes identified as lupeol, lupeyl acetate, lupeyl palmitate, taraxasterol, taraxasteryl acetate, pseudotaraxasterol, pseudotaraxasterol acetate, α-amyrin, α-amyryl palmitate, β-amyrin and β-amyryl palmitate, along with sitosterol, stigmasterol, palmitic and stearic acids. These compounds are been reported for the first time in the species. The compounds were identified by analysis of NMR spectra (¹H, 13C and DEPT), GC-MS and comparison with literature data. Previous work have reported the isolation of triterpenes, diterpenes, sesquiterpenes, flavonoids, coumarins and phenolic compounds from aerial parts and roots from G. polymorpha.

Gochnatia polymorpha subsp. floccosa; Asteraceae; triterpenes

Triterpenes from the flowers of Gochnatia polymorpha subsp. floccosa

Luana B. Silva^I; Regiane L. B. Strapasson^I; Dilamara Riva^I; Marcos J. Salvador^II; Maria Élida A. Stefanello^{I, *}

^IDepartamento de Química, Universidade Federal do Paraná, 81530-900 Curitiba-PR, Brazil

^IIInstituto de Biologia, Departamento de Biologia Vegetal, Curso de Farmácia, Universidade Estadual de Campinas, 13083-970 Campinas-SP, Brazil

ABSTRACT

Phytochemical study of the flowers of Gochnatia polymorpha subsp. floccosa, Asteraceae, yielded eleven known triterpenes identified as lupeol, lupeyl acetate, lupeyl palmitate, taraxasterol, taraxasteryl acetate, pseudotaraxasterol, pseudotaraxasterol acetate, α-amyrin, α-amyryl palmitate, β-amyrin and β-amyryl palmitate, along with sitosterol, stigmasterol, palmitic and stearic acids. These compounds are been reported for the first time in the species. The compounds were identified by analysis of NMR spectra (¹H, ¹³C and DEPT), GC-MS and comparison with literature data. Previous work have reported the isolation of triterpenes, diterpenes, sesquiterpenes, flavonoids, coumarins and phenolic compounds from aerial parts and roots from G. polymorpha.

Keywords:Gochnatia polymorpha subsp. floccosa; Asteraceae; triterpenes.

Introduction

Gochnatia polymorpha (Less.) Cabrera comprises three subspecies, with different distribution areas. The subspecies polymorpha is found in Southeastern of Brazil and Paraguay, the subspecies ceanothifolia is distributed in South of Brazil, Paraguay, Uruguay and Argentine, and the subspecies floccosa is limited to Southeast and South of Brazil (Cabrera & Klein, 1973). The genus Gochnatia is characterized by the production of sesquiterpene lactones, along with sesquiterpenes, diterpenes, triterpenes, acetylenes, flavonoids and coumarins (Catalan et al., 1996). Previous studies on G. polymorpha subsp. unspecified, reported the isolation of the triterpene bauerenyl acetate and dehydrocostus lactone from the bark and trunk bark (Farias et al., 1984), sesquiterpenes, sesquiterpene lactones and dimeric lactones from the roots and aerial parts (Bohlmann et al., 1986), flavonoids, phenolic compounds and a proline derivative from more polar extracts from leaves (Moreira et al., 2000), and essential oil of the wood (Lima et al., 2007). The studies on G. polymorpha subsp. polymorpha have reported the isolation of sesquiterpene lactones, diterpenes, triterpenes, flavonoids and coumarins from the roots and aerial parts (Sacilotto et al., 1997; Catalan et al., 2003). The essential oil composition and antimicrobial activity has been reported for Gochnatia polymorpha subsp. floccosa (Stefanello et al., 2006a, b), but there are no phytochemical investigations on this subspecies.

The present work describes, for the first time, the phytochemical study of the flowers of G. polymorpha subsp. floccosa.

Materials and Methods

NMR experiments were acquired on a Bruker AC-200 NMR, observing ¹H and ¹³C at 200 and 50 MHz, respectively. CDCl₃ was used as solvent and TMS as internal reference. The fatty acid methyl esters were analyzed by GC-EIMS (70 eV) using a Varian Saturn 2000 GC/MS spectrometer equipped with a CP-Sil-8CB capillary column (30 m x 0.25 mm, 0.25 µm film thickness), which was operated at 60 ºC for 3 min, and then programmed from 60-220 ºC at 5 ºC/min, after which it was kept isothermal at 220 ºC for 5 min. The carrier gas was helium (1 mL/min) and the injector temperature was 250 ºC. The compounds were identified by comparison of their retention indices (RI), calculated in relation to n-alkanes, and mass spectra with those found in the literature (Adams, 2007). Silica gel (Merck, 230-400 mesh) was used for column chromatographic separation (CC), while silica gel 60 PF₂₅₄ (Merck) was used for analytical (TLC, 0.25 mm) and preparative (PTLC, 1.0 mm) thyn layer chromatography. Compounds were visualized by exposure under UV_254/366 light and spraying with 5% (v/v) H₂SO₄ in ethanol solution, followed by heating on a hot plate.

Flowers of Gochnatia polymorpha subsp. floccosa Cabrera, Asteraceae, were collected on March 2004, in Curitiba, Paraná State, Brazil. The plant was identified by Dr. Armando Carlos Cervi and a voucher specimen was deposited in the herbarium of Universidade Federal do Paraná, in Curitiba, Paraná State, Brazil (UPCB 30100).

The air-dried flowers (126.8 g) were extracted exhaustively by maceration at room temperature with hexane and ethanol, successively. Crude ethanol extract (6.9 g), after removal solvent under reduced pressure, was suspended in EtOH:H₂O 1:1 and then extracted with CH₂Cl₂. The organic layer was separated and the solvent removed to give the residue (2.7 g). This was submitted to silica gel CC, eluted with increasing amounts of EtOAc in hexane to give eleven fractions. Fraction 2 (291.0 mg) was subjected to another CC eluted with hexane:EtOAC 95:5, yielding three subfrations. The subfraction 2.3 (102.8 mg) was purified by repeated PTLC eluted with pentane:EtOAc 95:5 to give M1 (13.6 mg, 2+5+7) and M2 (14.9 mg, 3+9+11). Fraction 6 (54.8 mg) was purified by repeated PTLC, eluted with hexane:CH₂Cl₂:EtOAc 2:1:0.5 to give M3 (7.2 mg, 1+4+6+8+10). Fraction 7 (49.1 mg) was purified by repeated PTLC eluted in hexane:CH₂Cl₂ 1:1 to give M4 (7.0 mg, palmitic + stearic acids). Fraction 8 (35.0 mg) was purified by PTLC eluted with hexane:EtOAc 8:2 to give M5 (12.2 mg, sitosterol + stigmasterol).

Results and Discussion

Chromatographic fractionation on silica gel of extracts rich in triterpenes commonly yields mixtures instead pure compounds. The identification of components can be achieved using standards and GC-MS analysis, or by careful analysis of NMR spectra of the mixture. The ¹H-NMR provides little information about structure of triterpenes, but ¹³C-NMR and DEPT are very useful. The most triterpenes found in vegetal extracts are pentacyclic or tetracyclic, with only one oxygenation on C-3 and one or two double linkage. In these cases, the type of the triterpene can be determined by chemical shifts and multiplicity of the carbons of the double linkage (Gallegos & Roque, 1990). This approach was applied in this work to identify the compounds found in the flowers of Gochnatia polymorpha subsp. floccosa.

The dichloromethane fraction of ethanol extract of flowers of G. polymorpha subsp. floccosa yielded mixtures (M1-M5) of triterpenes, steroids and fatty acids, which exhibited a single spot when analyzed by TLC in several solvents.

The ¹³C-NMR spectra of M1 showed sixty-seven signals, indicating a triterpene mixture. In particular was observed six signals for olefinic carbons, being two secondary (δ 107.1 and 109.4), one tertiary (δ 118.9) and three quaternary carbons (δ 139.8, 151.0 and 154.6). Comparison with literature data suggested the presence of triterpenes type lup-20(29)-ene (δ 109.4 and 151.0), urs-20(30)-ene (δ 107.1 and 154.6) and urs-20-ene (δ 118.9 and 139.8) (Gallegos & Roque, 1990). Also was observed an intense signal at δ 81.0, typical of an acetylated triterpene, and a signal for an ester carbonyl (δ 171.0). There were no others signals for oxygenated carbons. Thus the components of M1 were identified as lupeyl acetate (2), taraxasteryl acetate (5) and pseudotaraxasteryl acetate (7) (Gallegos & Roque, 1990; Mahato & Kundu, 1994; Silva et al., 1998) (Figure 1). The presence of acetates was corroborated by ¹H NMR that showed two singlets at δ 2.04 and 2.05. In addition also were observed signals for oxymethine (δ 4.48) and olefinic protons of 2 (δ 4.57 and 4.67), 5 (δ4.61) and 7 (δ5.26).

The ¹³C-NMR spectrum of M2 also showed more than sixty peaks, as M1. The signals of the olefinic carbons indicated the presence of three triterpenes type lup-20(29)-eno (δ 109.3 and 150.9), urs-12-ene (δ 124.3 and 139.6) and olean-12-ene (δ 121.6 and 145.2). The chemical shift of oxygenated carbon also was characteristic of an ester triterpene (δ 80.6), but the carbonyl group was more deshielded (δ 174.7) than in M1. In the ¹H NMR spectrum there were no signals for acetyl groups, but it was observed an intense signal at δ 1.25, typical of a long chain carbon. These data suggested the presence of triterpenes esterified with fatty acids. In order to identify the fatty acids, M2 was submitted to transesterification reaction with MeONa/MeOH (Mendes et al., 1999). After the usual work-up was obtained one fraction containing the sesquiterpene alcohols and another containing the fatty acid methyl esters. The last one was analyzed by GC-MS and showed only one compound (RI 1922), which was identified as methyl palmitate by its retention index, comparison with authentic sample and mass spectrum (Adams, 2007). Therefore, the components of M2 were identified as lupeyl palmitate (3), α-amyryl palmitate (9) and β-amyryl palmitate (11) (Figure 1). Mixtures with similar composition have been previously reported in Vernonia tweediana (Diaz et al., 2008) and Tapirira guianensis (Correia et al., 2008).

The ¹³C NMR spectra of M3 showed, in addition several signals for C-sp3, ten peaks for olefinic carbons, compatible with the presence of five triterpenes, type lup-20(29)-ene (δ 109.3 and 150.9), urs-20(30)-ene (δ 107.0 and 154.8), urs-20-ene (δ 118.9 and 139.5), urs-12-ene (δ 124.1 and 139.9) and olean-12-ene (δ 121.7 and 145.1). Also was observed a signal at δ 79.0, characteristic of oxymethine carbon of triterpene alcohol with the hydroxyl group β-orientated (Gallegos & Roque, 1990). This data suggested the presence of lupeol (1), taraxasterol (4), pseudotaraxasterol (6), α-amyrin (8) and β-amyrin (10) (Mahato & Kundu, 1994) (Figure 1). This composition was confirmed by ¹H NMR spectrum that showed signals for oxymethine protons (δ 3.22) and olefinic protons of 1 (δ 4.57 and 4.69), 4 (δ4.62), 6 (δ 5.26), 8 (δ5.17) and 10 (δ 5.13).

The mixture M4 showed NMR data typical of fatty acids. Esterification with MeOH yielded a mixture of palmitic and stearic methyl esters, which was identified by GC-MS based on their retention indices (1922 and 2122) and comparison of their mass spectra with literature (Adams, 2007) and with authentic sample.

The mixture M5 showed NMR data identical to those reported for the known mixture of the steroids sitosterol and stigmasterol (Goulart et al., 1993).

The identified triterpenes are been reported for the first time in G. polymorpha. The triterpene lupeol (1) was previously reported in the flowers of G. blanquetiana, along with sitosterol glycoside, kaempferol glycoside and 4-hydroxy-N-methylproline (Lima et al., 2003). Others phytochemical studies on flowers of Gochnatia species were not found.

Acknowledgements

This work was supported by Fundação Araucaria. The authors are grateful to Dr. Armando C. Cervi (Departamento de Botânica, UFPR) for plant identification. RLBS is grateful to CNPq for scholarship and MJS to FAPESP for financial support.

Received 14 Jul 2010

Accepted 23 Sep 2010

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Publication Dates

• Publication in this collection
  04 Mar 2011
• Date of issue
  June 2011

History

• Received
  14 July 2010
• Accepted
  23 Sept 2010