Terpenoids, flavonoids and other constituents of Eupatorium betonicaeforme (Asteraceae)

Abstracts

A new acylated kaurene diterpene, characterized as 15alpha-decanoyloxy-kaur-16-en-19-oic acid, along with nine known compounds: pentacosanoic acid, 24alpha-ethyl-5alpha-cholesta-7,22E-dien-3beta-ol, 15alpha-hydroxy-kaur-16-en-19-oic acid, 8beta-angeloyloxy-9beta,10beta-dihydroxy-1-oxo-germacra-4E,11(13)dien-12,6alpha-olide, 3beta-hydroxyeicosan-1,5beta-olide, taraxasteryl acetate, 7-O-methylkaempferol, kaempferol, and nepetin were isolated from the flowers of Eupatorium betonicaeforme (Asteraceae). In addition, from the aerial parts were isolated taraxasteryl acetate and alpha- and beta-amyrin, while the mixture of beta-sitosterol and stigmasterol, and 6-acetyl-2,2-dimethylchroman-4-one were isolated from the roots. The structure elucidation of all compounds was performed by spectroscopic analysis and comparison with published data from literature.

Eupatorium betonicaeforme; Asteraceae; triterpenes; diterpenes; flavonoids; lactones

Um novo diterpeno kaureno, caracterizado como ácido 15alfa-decanoiloxi-kaur-16-en-19-óico, juntamente com os compostos conhecidos: ácido pentacosanóico, 24alfa-etil-5alfa-colesta-7,22E-dien-3beta-ol, ácido 15alfa-hidroxi-kaur-16-en-19-óico, 8beta-angeloil-9beta,10beta-diidroxi-1-oxo-germacra-4E ,11(13)dien-12,6alfa-olideo, 3beta-hidroxiicosan-1,5beta-olideo, acetate de taraxasterila, 7-O-metilcampferol, caempferol e nepetina foram isolados das flores de Eupatorium betonicaeforme. Adicionalmente, foram isolados das partes aéreas, o acetato de taraxasterila e a mistura binária de alfa- e beta-amirina, enquanto foram obtidos das raízes, a mistura de beta-sitosterol e estigmasterol, e 6-acetil-2,2-dimetilcroman-4-ona. A elucidação estrutural de todos os compostos isolados neste trabalho foi realizada por análise espectroscópica e comparação com dados descritos na literatura.

ARTICLE

Terpenoids, flavonoids and other constituents of Eupatorium betonicaeforme (Asteraceae)

Maria Rose Jane R. Albuquerque; Andreza Maria L. Pires; Otília Deusdênia L. Pessoa^*; Edilberto R. Silveira

Curso de Pós-Graduação em Química Orgânica, Departamento de Química Orgânica e Inorgânica, Universidade Federal do Ceará, CP 12200, 60021-970 Fortaleza - CE, Brazil

ABSTRACT

A new acylated kaurene diterpene, characterized as 15a-decanoyloxy-kaur-16-en-19-oic acid, along with nine known compounds: pentacosanoic acid, 24a-ethyl-5a-cholesta-7,22E-dien-3b-ol, 15a-hydroxy-kaur-16-en-19-oic acid, 8b-angeloyloxy-9b,10b-dihydroxy-1-oxo-germacra-4E,11(13)dien-12,6a-olide, 3b-hydroxyeicosan-1,5b-olide, taraxasteryl acetate, 7-O-methylkaempferol, kaempferol, and nepetin were isolated from the flowers of Eupatorium betonicaeforme (Asteraceae). In addition, from the aerial parts were isolated taraxasteryl acetate and a- and b-amyrin, while the mixture of b-sitosterol and stigmasterol, and 6-acetyl-2,2-dimethylchroman-4-one were isolated from the roots. The structure elucidation of all compounds was performed by spectroscopic analysis and comparison with published data from literature.

Keywords:Eupatorium betonicaeforme, Asteraceae, triterpenes, diterpenes, flavonoids, lactones

RESUMO

Um novo diterpeno kaureno, caracterizado como ácido 15a-decanoiloxi-kaur-16-en-19-óico, juntamente com os compostos conhecidos: ácido pentacosanóico, 24a-etil-5a-colesta-7,22E-dien-3b-ol, ácido 15a-hidroxi-kaur-16-en-19-óico, 8b-angeloil-9b,10b-diidroxi-1-oxo-germacra-4E ,11(13)dien-12,6a-olideo, 3b-hidroxiicosan-1,5b-olideo, acetate de taraxasterila, 7-O-metilcampferol, caempferol e nepetina foram isolados das flores de Eupatorium betonicaeforme. Adicionalmente, foram isolados das partes aéreas, o acetato de taraxasterila e a mistura binária de a- e b-amirina, enquanto foram obtidos das raízes, a mistura de b-sitosterol e estigmasterol, e 6-acetil-2,2-dimetilcroman-4-ona. A elucidação estrutural de todos os compostos isolados neste trabalho foi realizada por análise espectroscópica e comparação com dados descritos na literatura.

Introduction

The genus Eupatorium (family Asteraceae, tribe Eupatorieae, subtribe Eupatoriinae), is a taxonomically complex group of species with distribution centers mainly in Europe, eastern Asia and North America.¹ Several studies on Eupatorium species have been reported, revealing a great number and diversity of secondary metabolites, where the sesquiterpenes lactones of the guaianolide,² germacranolide,³ heliangolide,⁴ and eudesmanolide types⁵ are the most characteristic compounds. Flavonoids,⁶ diterpenes,⁷ benzofurans,⁸ pyrrolizidine alkaloids,⁹ chromenes¹⁰ and thymol derivatives¹¹ are also common. Triterpenes,¹² sesquiterpenes,¹³ coumarins¹⁴ and d-lactones of the long chain type¹⁵ have been occasionally isolated. A great number of Eupatorium species are employed in traditional medicine in the treatment of different pathologies. Indeed, a significant number of bioactive natural compounds have been reported from this renowned genus.^16-19

As part of a continuing search on native plants from Northeast of Brazil, particularly of the Asteraceae family, the volatile oils from the roots and leaves of E. betonicaeforme yielded 2,2-dimethyl-6-vinylchroman-4-one (1) and 2-senecioyl-4-vinylphenol (2), the sesquiterpenes b-caryophyllene, g-muurolene and bicyclogermacrene as the main constituents. Both oils showed larvicidal effects against Aedes aegypti larvae, particularly the oil from the roots.²⁰ The major constituents (1 and 2) were isolated and tested. Compound 1 was the most active compound and is probably responsible for the larvicidal effect.²⁰ Continuing with the phytochemical investigation of E. betonicaeforme this paper reports now its non-volatile chemical composition.

Results and Discussion

The extract of the flowers of E. betonicaeforme, after successive column chromatography on Si gel, afforded the new secondary metabolite 15a-decanoyloxy-kaur-16-en-19-oic acid (3), together with the known compounds: pentacosanoic acid (4), 24a-ethyl-5a-cholesta-7,22E-dien-3b-ol (5),²¹ 15a-hydroxy-kaur-16-en-19-oic acid (6),²² 8b-angeloyloxy-9b,10b-dihydroxy-1-oxo-germacra-4E,11(13)dien-12,6a-olide (7),²³ 3b-hydroxyeicosan-1,5b-olide (8),²⁴ taraxasteryl acetate (9),²⁵ 7-O-methylkaempferol (10), kaempferol (11), and nepetin (12).²⁶ From the hexane extract of the aerial parts (leaves and stems) were isolated taraxasteryl acetate (9) and a- and b-amyrin (13 and 14),²⁵ while from the hexane extract of roots were obtained a mixture of b-sitosterol and stigmasterol (15 and 16) and 6-acetyl-2,2-dimethylchroman-4-one (17),²⁷Figure 1. All of the known compounds were identified by comparison of their spectral data with those reported in the literature. Compound 7 was previously isolated from Trichogonia salviaefolia (Asteraceae) by Bohlmann et al., and its molecular structure has been deduced by ¹H NMR and MS.²³ Complete ¹H and ¹³C NMR chemical shifts of 7, which were assigned by a combination of 1D and 2D NMR techniques, are listed in Table 1.

Compound 3 was isolated as a colorless waxy solid. The molecular formula C₃₀H₄₈O₄ was deduced by a combination of EIMS ([M]⁺, m/z 472) and ¹³C NMR data. The ¹³C NMR spectrum showed a total of 30 carbon signals, among the characteristic signals at d_C 29.9-29.3 and 14.3 consistent with the presence of aliphatic methylene groups of a fatty acyl moiety. The deshielded carbon signals at d_C 184.8 and 174.1 indicated the presence of two carbonyl groups compatible with the presence of carboxylic acid and ester functions, respectively. IR absorption bands at 1730 and 1694 cm^-1 further supported these findings. The additional deshielded carbon signals at d_C at 155.7 and 110.0, as well the proton signal at d_H 5.26 (2H, brs) clearly demonstrated the presence of an exomethylene moiety. In the HMBC spectrum were observed correlations for the exocyclic double-bond protons (d_H 5.26) with the oxymethine carbon signal at d_C 82.9 revealing the contiguity of these units. Comparison of the chemical shifts with the literature data indicated the presence of a kaur-16-en-19-oic acid bearing an oxygen atom at C-15.^22,28 This fact was also reinforced by the EIMS, which showed an intense ion peak at m/z 318 (C₂₀H₃₀O₃) related to a proton rearrangment followed by loss of the fatty side chain. In accordance with published data, it was assumed that diterpene 3 belongs to the ent-series, like the other kaurenes previously isolated from Eupatorium.^28,29 Additional support for this affirmation was the small negative specific rotation ( -0.66°, c 0.05, CHCl₃), which is characteristic of ent-kaurenes.³⁰ The location of the acyl moiety was supported by the long-range correlation of the oxymethine proton at d_H 5.26 (H-15) with the ester carbonyl signal at d_C 174.1, while the a-orientation of this unit was inferred by comparison of the ¹³C NMR data with those from cinannamoylgrandifloric acid.²² Thus, the structure of 3, a new ent-kaurene derivative, was established as 15a-decanoyloxy-16-kauren-19-oic acid. Although the labdanes are the most frequently encountered diterpenes in plants of the genus Eupatorium, ent-kauranes are not uncommon.^28,29

Experimental

General experimental procedures

Melting points were determined using a digital Mettler Toledo FP90 apparatus. The optical rotations were measured on a Perkin-Elmer 341 digital polarimeter. IR spectra were recorded using a Perkin-Elmer FT-IR 1000 spectrometer. EIMS was acquired with the direct insertion probe on a GC-MS Shimadzu spectrometer at 70 eV. NMR spectra were recorded on a Bruker Avance DRX-500 (500 MHz for ¹H and 125 MHz for ¹³C) or DPX-300 (300 MHz for ¹H and 75 MHz for ¹³C) spectrometers. Chemical shifts, given on the d scale, were referenced to the residual undeuterated portion of the deuterated organic solvent, for proton [(CD₃)₂CO: d_H 2.05; CDCl₃: d_H 7.27], and the center peak of the deuterated solvent [(CD₃)₂CO: d_C 206.68, 29.92; CDCl₃: d_C 77.23]. Column chromatography was run using silica gel 60 (70 - 230 mesh, VETEC) and silica gel 60 (0.063-0.200 mm, MERCK). TLC was performed on precoated silica gel polyester sheets (Kieselgel 60 F₂₅₄, 0.20 mm, Merck) by detection with a spray reagent of vanillin/perchloric acid/EtOH solution followed by heating at 120 °C.

Plant material

Eupatorium betonicaeforme in the flowering stage was collected in May 2002, from Acarape County, Ceará, and identified by Prof. Elnatan B. Souza of the Universidade Estadual Vale do Acaraú - Ceará. Voucher specimen (# 29.104) has been deposited at the Herbario Prisco Bezerra (EAC) of the Departamento de Biologia, Universidade Federal do Ceará.

Extraction and isolation

Air-dried and powdered E. betonicaeforme parts were extracted with hexane: flowers (0.8 kg, 2 ´ 4 L), aerial parts (2.5 kg, 2 ´ 9 L) and roots (0.7 kg, 2 ´ 3 L), followed by EtOH with same amounts of solvent. After evaporation of the solvents under reduced pressure afforded the crude hexane and EtOH extracts, respectively: flowers (15.9 g and 82.9 g), aerial parts (51.6 g and 75.9 g), and roots (4.6 g and 18.0 g). The hexane extract from the flowers (15.9 g) was fractioned over Si gel to yield the follow fractions: hexane (0.4 g), hexane/EtOAc 8:2 (14.0 g), hexane/EtOAc 1:1 (0.8 g) and EtOAc (0.3 g). The hexane/EtOAc fraction 8:2 (14.0 g) was chomatographed over Si gel, eluting with hexane/EtOAc (9:1, 1:1), EtOAc, and MeOH to afford four fractions (F1-F4). F4 (8.7 g, hexane/EtOAc 9:1) was subjected to successive Si gel flash column chromatography using hexane/EtOAc 9.5:0.5 to yield 3 (80.0 mg) and pentacosanoic acid 4 (12.0 mg). Similarly, F2 (1.7 g, hexane/EtOAc 1:1) was subjected to repeated Si gel flash column chromatography using hexane/EtOAc 9:1 mixture to yield 5 (13.0 mg) and 6 (5.8 mg). The hexane/EtOAc fraction 1:1 (0.8 g) was subjected to Si gel flash column chromatography using CHCl₃/EtOAc 9:1 to give 145 subfractions of 10 mL each. 7 (16.0 mg) was isolated from fractions 65-72, while 8 (21.0 mg) was obtained from fractions 89-105. The EtOH extract of the flowers (82.0 g) was suspended in MeOH/H₂O 3:1 and partitioned with CH₂Cl₂, EtOAc, and n-BuOH. The CH₂Cl₂ extract was evaporated under reduced pressure yielding a residue (24.0 g), which was fractioned over Si gel and eluted with hexane/EtOAc (7.5:2.5, 1:1, 2.5:7.5) and EtOAc. The hexane/EtOAc (7.5:2.5) fraction (4.0 g) was chromatographed over Si gel eluting with hexane/EtOAc (9:1, 8:2, 7:3, 1:1), EtOAc and MeOH to afford seven fractions (F1-F7). F2 (1.2 g) was subjected to Si gel flash column chromatography using hexane/EtOAc (9.9:0.1) to afford 9 (40.0 mg), while from fraction F6 (0.1 g), using the same chromatographic procedure and the solvent system hexane/EtOAc (7:3) was isolated 10 (3.0 mg). The hexane/EtOAc (1:1) fraction (1.1 g) was subjected to Si gel flash column chromatography eluting with a CHCl₃/EtOAc (8:2) gradient to yield 11 (4.2 mg) and 12 (370.0 mg). The hexane extract from the aerial parts (51.0 g) was fractioned over Si gel eluting with hexane, CHCl₃ and EtOAc. The hexane fraction (25.0 g) was chromatographed over Si gel using petroleum ether, CH₂Cl₂ and EtOAc as eluent. An aliquot of the petroleum ether fraction (1.5 g) was subjected to hydrolysis with KOH solution to yield a free fatty acids mixture (0.07 g) and an unsaponifiable fraction (0.7 g). Methylation of the fatty acids with MeOH/HCl (1.0%) followed by GC/MS analysis of the methyl esters mixture allowed the identification of: hexadecanoic (65.0%), heptadecanoic (1.2%), octadecanoic (7.3%), eicosanoic (3.5%), docosanoic (9.0%), tricosanoic (1.0%), and tetracosanoic acids (3.5%). TLC analysis indicated that the CH₂Cl₂ fraction (9.8 g) was rich in compound 9, which was recrystallized in acetone to afford taraxasteryl acetate (736.0 mg). The CH₂Cl₂ fraction (21.0 g) after successive Si gel column chromatography using hexane and increasing amounts of EtOAc afforded a binary mixture of 13 and 14 (7.0 mg). The hexane fraction from the roots (4.6 g) was fractioned over Si gel eluting with hexane, hexane/CH₂Cl₂ 1:1, CH₂Cl₂ and EtOAc. From the fraction hexane/CH₂Cl₂ (1:1) fraction (1.6 g) was obtained a mixture of 15 and 16 (30.0 mg). The EtOH extract was fractioned over Si gel eluting with hexane/EtOAc (8:2, 6:4, 4:6, 2:8), EtOAc and MeOH. The hexane/EtOAc (8:2) fraction (2.7 g) was subjected to successive Si gel column chromatography using increasing amounts of EtOAc in hexane to yield 17 (42.0 mg).

15a-Decanoyloxy-kaur-16-en-19-oic acid (3). Colorless waxy solid; -0.66° (c 0.05, CHCl₃); IR (KBr) n_max/cm^-1: 3350-2700, 1730, 1694, 1464, 1242, 1165; EIMS (70 eV) m/z 472 [M]⁺ (5), 318 (25), 300 (80), 285 (90), 255 (50), 104 (65), 91 (45), 71 (82), 57 (100); ¹H (500 MHz) and ¹³C NMR (125 MHz) data, Table 1.

8b-Angeloyl-9b,10b-dihydroxy-1-oxo-germacra -4E,11(13)dien-12,6a-olide (7). Colorless needles; mp 164-165 °C; -0.24° (c 0.05, acetone); IR (KBr) n_max/cm^-1: 3437, 2929, 1736, 1699, 1154, 1107; EIMS m/z 378 [M]⁺ (absent), 312 (10), 295 (5), 277 (15), 260 (12), 232 (20), 207 (12), 108 (13), 83 (100); ¹H (500 MHz) and ¹³C NMR (125 MHz) data, Table 1.

Acknowledgments

The authors acknowledge the financial support provided by the Brazilian Agencies CNPq, CAPES, FUNCAP and PRONEX.

Received: June 6, 2005

Published on the web: November 18. 2005

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Publication Dates

• Publication in this collection
  14 Mar 2006
• Date of issue
  Feb 2006

History

• Accepted
  18 Nov 2005
• Received
  06 June 2005