Chemical constituents of the underground stem bark of Duguetia furfuracea (Annonaceae)

Abstracts

In the present investigation the underground parts of Duguetia furfuracea (Annonaceae) were used to conduct a phytochemical study that included the brine shrimp (Artemia salina) lethality bioassay. The substances (-)-duguetine beta-N-oxide, (-)-duguetine, dicentrinone, (-)-N-methyltetrahydropalmatine, and (+)-N-methylglaucine were isolated from the alkaloid extract of the bark of the underground stem, and the ureide allantoin was also isolated by precipitation from the ethanol extract of the wood of the underground stem. A fresh volatile oil and a nonpolar extract were also obtained from the underground stem bark. The substances 2,4,5-trimethoxystyrene, a-gurjunene, aromadendrene, bicyclogermacrene, (E)-methylisoeugenol, and alpha-asarone were isolated from the fresh volatile oil and polycarpol, beta-caryophyllene oxide, 2,4,5-trimethoxystyrene, a-asarone, and asaraldehyde were obtained from the petroleum ether extract. The present study describes for the first time the alkaloid (-)-duguetine beta-N-oxide and the occurrence of (-)-N-methyltetrahydropalmatine and (+)-N-methylglaucine in the family Annonaceae. All extracts were active in the brine shrimp lethality bioassay.

alkaloids; Annonaceae; aporphines; duguetine beta-N-oxide; Duguetia furfuracea; sesquiterpenoids

No presente trabalho foi realizado um estudo fitoquímico com os diferentes extratos obtidos a partir de órgãos subterrâneos de Duguetia furfuracea (Annonaceae), o qual incluiu o teste de toxicidade para Artemia salina. O extrato alcaloídico, obtido das cascas do caule subterrâneo, conduziu ao isolamento de (-)-duguetina beta-N-óxido, de (-)-duguetina, dicentrinona, (-)-N-metiltetraidropalmatina e (+)-N-metilglaucina. Do extrato etanólico do cerne do caule subterrâneo obteve-se alantoína por precipitação. O óleo volátil e o extrato apolar também foram extraídos das cascas do caule subterrâneo. As substâncias 2,4,5-trimetoxiestireno, alfa-gurjuneno, aromadendreno, biciclogermacreno, (E)-metil-isoeugenol e a-asarona foram isoladas a partir do óleo volátil, e as substâncias policarpol, óxido de beta-cariofileno, 2,4,5-trimetóxi-estireno, alfa-asarona e asaraldeído foram obtidas do extrato em éter de petróleo. Este estudo descreve pela primeira vez o alcalóide beta-N-óxido de (-)-duguetina, e a ocorrência das substâncias (-)-N-metiltetraidropalmatina e (+)-N-metilglaucina na família Annonaceae. Todos os extratos se mostraram tóxicos nos testes com Artemia salina.

ARTICLE

Chemical constituents of the underground stem bark of Duguetia furfuracea (Annonaceae)

Denise B. da Silva^I; Elaine C. O. Tulli^I; Walmir S. Garcez^II; Evandro A. Nascimento^III; João M. de Siqueira^{I, *}

^IDepartamento de Farmácia, Universidade Federal do Mato Grosso do Sul, CP 579, 79070-900 Campo Grande-MS, Brazil

^IIDepartamento de Química, Universidade Federal do Mato Grosso do Sul, CP 579, 79070-900 Campo Grande-MS, Brazil

^IIIInstituto de Química, Universidade Federal de Uberlândia, Av. João Naves de Ávila, 121, Campus Santa Mônica, CP 593, 38400-089 Uberlândia-MG, Brazil

ABSTRACT

In the present investigation the underground parts of Duguetia furfuracea (Annonaceae) were used to conduct a phytochemical study that included the brine shrimp (Artemia salina) lethality bioassay. The substances (-)-duguetine b-N-oxide, (-)-duguetine, dicentrinone, (-)-N-methyltetrahydropalmatine, and (+)-N-methylglaucine were isolated from the alkaloid extract of the bark of the underground stem, and the ureide allantoin was also isolated by precipitation from the ethanol extract of the wood of the underground stem. A fresh volatile oil and a nonpolar extract were also obtained from the underground stem bark. The substances 2,4,5-trimethoxystyrene, a-gurjunene, aromadendrene, bicyclogermacrene, (E)-methylisoeugenol, and a-asarone were isolated from the fresh volatile oil and polycarpol, b-caryophyllene oxide, 2,4,5-trimethoxystyrene, a-asarone, and asaraldehyde were obtained from the petroleum ether extract. The present study describes for the first time the alkaloid (-)-duguetine b-N-oxide and the occurrence of (-)-N-methyltetrahydropalmatine and (+)-N-methylglaucine in the family Annonaceae. All extracts were active in the brine shrimp lethality bioassay.

Keywords: alkaloids, Annonaceae, aporphines, duguetine b-N-oxide, Duguetia furfuracea, sesquiterpenoids

RESUMO

No presente trabalho foi realizado um estudo fitoquímico com os diferentes extratos obtidos a partir de órgãos subterrâneos de Duguetia furfuracea (Annonaceae), o qual incluiu o teste de toxicidade para Artemia salina. O extrato alcaloídico, obtido das cascas do caule subterrâneo, conduziu ao isolamento de (-)-duguetina b-N-óxido, de (-)-duguetina, dicentrinona, (-)-N-metiltetraidropalmatina e (+)-N-metilglaucina. Do extrato etanólico do cerne do caule subterrâneo obteve-se alantoína por precipitação. O óleo volátil e o extrato apolar também foram extraídos das cascas do caule subterrâneo. As substâncias 2,4,5-trimetoxiestireno, a-gurjuneno, aromadendreno, biciclogermacreno, (E)-metil-isoeugenol e a-asarona foram isoladas a partir do óleo volátil, e as substâncias policarpol, óxido de b-cariofileno, 2,4,5-trimetóxi-estireno, a-asarona e asaraldeído foram obtidas do extrato em éter de petróleo. Este estudo descreve pela primeira vez o alcalóide b-N-óxido de (-)-duguetina, e a ocorrência das substâncias (-)-N-metiltetraidropalmatina e (+)-N-metilglaucina na família Annonaceae. Todos os extratos se mostraram tóxicos nos testes com Artemia salina.

Introduction

Nearly 80 species are known in the genus Duguetia, which is one of the 128 genera included in the family Annonaceae.¹

This paper reports the results of our continued phytochemical investigations of the family Annonaceae. We analyzed various extracts obtained from the underground stem bark of Duguetia furfuracea (A. St.-Hil.) Benth. & Hook f., which is a shrub distributed throughout the Brazilian state of Mato Grosso do Sul. Two species of the genus Duguetia have been found and worked up to date, and little data on their use in folk medicine can be found in the literature.

D. furfuracea is known as "araticum-seco".² In folk medicine, the seed powder is mixed with water for use in the treatment of pediculosis,³ while an infusion of the leaves and twigs is used to treat rheumatism, and a medicine derived from this plant is claimed to be useful in the treatment of renal colic.⁴ On the other hand, this species is a major invader of pastures and therefore is harmful to the regional economy, which is based predominantly on agribusiness. Thus, the chemical knowledge of the species could contribute to control its weedy behaviour.

The chemical constituents of the aerial parts (leaves and twigs) of D. furfuracea are described in three previous papers.^5-7 The first paper describes the isolation of sesquiterpenoids;⁵ the other two papers report the isolation of a flavonoid and various alkaloids from an isoquinoline-derived skeleton,⁶ and the trypanocidal activity of some of these compounds. One of these papers⁷ describes two aporphine alkaloids containing N-nitroso functionality.

The present investigation, which adds to the knowledge of this species, led to the isolation of alkaloids, sesquiterpenoids and other constituents from different extracts obtained from the underground stem bark of D. furfuracea.

An alkaloid extract was obtained from the dried and finely ground stem bark, from which (-)-duguetine b-N-oxide (1), (-)-duguetine (2), dicentrinone (3), (-)-N-methyltetrahydropalmatine (4), and (+)-N-methylglaucine (5) were isolated. Also, the ureide allantoin (6) was isolated from the ethanol extract of the underground stem (Figure 1).

The substances 2,4,5-trimethoxystyrene (7), a-gurjunene (8), aromadendrene (9), bicyclogermacrene (10), (E)-methylisoeugenol (11), and a-asarone (12) were isolated from the fresh volatile oil. The substances polycarpol (13), b-caryophyllene oxide (14), asaraldehyde (15), 7 and 12 were obtained from the petroleum ether extract (Figure 1).

The ¹H and ¹³C NMR data of 1, including 2D NMR data (NOESY, HMQC and HMBC) and (+) HR-ESIMS characterized this compound as an aporphine alkaloid. To the best of our knowledge, this compound has not been previously reported in the literature. For the first time, substances 6 and 12 are described in the genus Duguetia and substances 4 and 5, in the family Annonaceae.

Results and Discussion

The alkaloid extract was fractionated by column chromatography (CC), yielding a novel alkaloid identified as (-)-duguetine b-N-oxide (1), and four other known alkaloids (2, 3, 4, 5).

Compound 1 was obtained as a brown amorphous solid and exhibited –33.3 (MeOH, c 0.001). Its molecular formula (C₂₀H₂₁NO₆) was determined using HR-ESIMS (m/z 372.1413, [M+H]⁺). Its FTIR spectrum revealed the presence of a hydroxyl group (3418 cm^-1) and an aliphatic system (2959-2846 cm^-1). The ¹H NMR data showed three hydrogen signals in the aromatic region at d 7.53 (s), 7.30 (s), and 6.47 (s) and an N-linked methyl group at d 3.24 (s).

The presence of 1,2-methylenedioxy and 9,10-dimethoxy groups was indicated in the NMR spectrum by two singlet signals at d 3.84 (s) and 3.90 (s), and the typical pair of doublets of methylenedioxy protons was observed at d 6.10 and 5.93 (d, 1.1 Hz). Also, were observed aliphatic protons signals at d 3.59 (m) and 2.91 (m). These ¹H NMR data (Table 1) are in agreement with an aporphine alkaloid having 1,2-methylenedioxy and 7-hydroxy groups with trans configuration at 6a,7, exhibiting a large coupling constant for the two doublets at d 5.08 (d, 12.1 Hz) and 4.34 (d, 12.1 Hz).

The ¹³C NMR data were also in agreement with the observations (Table 1), but the chemical shift of carbon atoms in the neighborhood of the N-heterocycle suggests the presence of an N-O group, as the N-heterocycle region showed a more pronounced deshielding effect than in duguetine (2) (Figure 2).

This effect is in accordance with the molecular formula established by HR-MS, indicating the presence of an additional oxygen atom.⁸ By comparing the ¹³C NMR data of 1 with those of 2, using N-O models available in the literature, namely oliveroline b-N-oxide (6a,7a-H, b-N-oxide)⁹ and dasymaschaline a-N-oxide (6a,7a-H, a-N-oxide),⁸ and confirming with NOESY the stereochemistry of the N-oxide moiety of 1, it was possible to propose that 1 is a (-)-duguetine b-N-oxide (Table 1).

Allantoin (6) was isolated from the ethanol extract by precipitation with acetone and its spectral data were agree with those in the literature.¹⁰ This metabolite belongs to the ureide class and plays an important role in the transport and assimilation in some nitrogen-fixing species.¹¹ Allantoin, which is also used in cosmetics,¹² exhibits anti-inflammatory activity among other properties. This compound has already been obtained in large amounts from the underground parts of other pasture-invading species.¹⁰

The fresh volatile oil was fractionated by CC on silica gel, yielding several fractions. The fractions, composed of a mixture of two compounds, were subjected to an additional preparative thin layer chromatography (TLC) followed by preparative argentation TLC separation, and six compounds (7-12) were isolated and identified by comparison of their spectral data with literature values (see Experimental section). In order to contribute to the chemical knowledge of nonpolar compounds from the fresh stem bark, the fresh volatile oil was subjected to GC/MS analysis. The compounds identified by this method are listed in Table 2.

In a further chemical investigation of the fresh stem bark, the petroleum ether extract yielded, by the usual phytochemical work up, 7, 12, 13, 14 and 15 (Figure 1).

Volatile and nonpolar compounds have been described in Annonaceae.¹³ Essential oils, for instance, are responsible for the fragrance of several species and their chemical composition generally includes well-known monoterpenes, sesquiterpenes, or aromatic compounds.¹⁴

A noteworthy feature is the fact that the isolation of aromatic compounds such as propenylbenzenes (or vinylbenzenes) has been described predominantly in the underground parts.^14,15 Phenylpropanoid-derived compounds are widespread among vascular plants and play an important role in chemical defense.¹⁶

With regard to polycarpol (13), it has been described as a chemical marker of the family Annonaceae, however it has recently been found in another plant family.¹⁷

The fresh volatile oil and the petroleum ether and alkaloid extracts were active in the brine shrimp lethality bioassay, with LD₅₀ values of 2.6, 6.1, and 36.9 mg mL^-1, respectively.

The toxicity exhibited by the extracts and fresh volatile oil might be explained by the presence of active substances previously tested in the brine shrimp lethality bioassay, namely 2,4,5-trimethoxystyrene (7) (LD₅₀ = 8 µg mL^-1),¹⁸ found in both the fresh volatile oil and petroleum ether extract, polycarpol (13) (LD₅₀ = 254 µg mL^-1),¹⁹ in the petroleum ether extract, and several oxoaporphine alkaloids.²⁰

Experimental

General experimental procedures

Optical rotations were measured on a Perkin-Elmer 341 polarimeter in MeOH. The FTIR spectra of either KBr pellets or CHCl₃ films were obtained on a Perkin Elmer 783 spectrophotometer. The MS system (HR-ESIMS) used was a quadrupole time-of-flight instrument (UltrOTOF-Q, Bruker Daltonics, Billerica, MA), equipped with an ESI source; the analyses were performed with the mass spectrometer in the positive ion mode. The GC/MS data were obtained on a CG17A/QP5000 Shimadzu GC/MS instrument equipped with a DB-5 capillary column using He as the carrier gas. The ¹H and ¹³C 1D and 2D NMR spectra were recorded at 300 MHz (¹H) and 75 MHz (¹³C) on a Bruker DPX-300 spectrometer.

Plant material

The underground parts (stem bark and wood) of Duguetia furfuracea (A. St.-Hil.) Benth. & Hook f. were collected in March 2004, on the UFMS campus in Campo Grande, MS, Brazil, and identified by Prof. R. Mello-Silva. A voucher specimen (No. 023) was deposited in the CGMS Herbarium (UFMS, Campo Grande, MS).

Extraction and isolation of compounds

Ammonium hydroxide was added to 1300 g of air-dried and finely ground bark of the underground stem until pH 9.0 was reached and then subjected to exhaustive extraction with CHCl₃. The resulting chloroform layer was concentrated under reduced pressure and a brown residue (68 g) was obtained.

The residue was solubilized in CHCl₃ and extracted with 5% HCl; the resulting acid fraction was adjusted to pH 9.0 (NH₄OH) and again exhaustively extracted with CHCl₃.

A brown precipitate was obtained (9.2 g). This extract (7.2 g) was fractionated by CC on alumina (70-230 mesh) and eluted with CHCl₃:MeOH:H₂O gradient system to yield thirteen fractions (I-XIII).

Fractions IV, X and XI from this column provided 2 (812.1 mg), 4 (69.3 mg) and 5 (11.2 mg). Fraction V (800 mg) was fractionated by CC on silica gel (70-230 mesh) in CHCl₃:MeOH:H₂O. Fractions 60-75 yielded 3 (4.6 mg). Fraction VII (378.9 mg) yielded 1 after CC on silica gel (70-230 mesh) eluted with CHCl₃:EtOAc:MeOH:H₂O gradient system.

From the ethanol extract of the underground stem, the ureide allantoin (6) was isolated by precipitation with acetone.

The alkaloids 2, 3, 4 and 5, as well as the compounds 6-12 were identified by ¹H and ¹³C NMR spectroscopy and compared with literature values.^10,18-31

(-)-Duguetine b-N-oxide, (1)

Brown amorphous solid; Rf 0.51 (CHCl3:MeOH, 85.15); : –33.3º (MeOH; c 0.001,); FTIR (KBr) n_max/cm^-1: 3418, 2959, 2929, 2846, 1607, 1515, 1463, 1393, 1340, 1244, 1226, 1217, 1118, 1046, 993, 945, 870; (+) HR-ESIMS m/z: 372.1413 [M+H]⁺ (Calc. for C₂₀H₂₂NO₆ requires 372.1436). ¹H NMR (300 MHz, CDCl₃, d) and ¹³C NMR (75 MHz, CDCl₃, d): see Table 1.

(-)-N-Methyltetrahydropalmatine, (4)

Brown amorphous solid; : –104.1º (MeOH; c 0.0012). FTIR, ¹H and ¹³ C NMR data are in agreement with those reported in the literature.³⁰

(+)-N-Methylglaucine, (5)

Brown amorphous solid; : +34.9º (MeOH; c 0.0012). FTIR, ¹H and ¹³C NMR data are in agreement with those reported in the literature.³¹ A 4-hour hydrodistillation of 300 g of fresh bark of underground stem, using a Clevenger-type apparatus, yielded 1.2% fresh volatile oil.

The oil was fractionated by CC on silica gel (70-230 mesh), and eluted with petroleum ether:CHCl₃:MeOH gradient system to yield thirteen combined fractions (I-XIII). Compound 7 (117.1 mg) was identified as the major component of fraction XII, and compound 12 (160.2 mg), of fraction XIII. Fractions II and XI from this column provided 8 (19.5 mg) and 11 (10.1 mg), respectively. Fractions III and VI were purified by preparative/argentation TLC yielding 9 (9.1 mg) and 10 (17.2 mg), respectively.

A separate amount of air-dried and finely ground bark of the underground stem (155 g) was subjected to exhaustive petroleum ether extraction in a Soxhlet apparatus; the resulting extract was concentrated in vacuo to dryness (13 g). The precipitate from this extract was purified by recrystallization from hexane and CHCl₃ providing 13 (176.4 mg). The resulting extract (5 g) was then fractionated by CC on silica gel (70-230 mesh) using hexane:CHCl₃:MeOH as elution gradient. The fractions showing similar spots on TLC were combined into twelve fractions (I-XII). Fractions I and IX provided 14 (11.8 mg) and 15 (12.3 mg), respectively. Fraction X (235.2 mg) yielded a mixture of 7 and 12.

Brine shrimp lethality test

The brine shrimp (A. salina Leach) toxicity tests were conducted using second instar larvae according to the method of McLaughlin. Stock solutions of samples were prepared by dissolving 15 mg of the test material in 5 mL of sea water containing 1% (v/v) DMSO. The assays were carried out in triplicate on samples at a concentration of 500, 50, 5.0 and 0.5 µg mL^-1. Positive (Quinidine Sulfate) and negative (sea water containing 0.1% (v/v) DMSO) controls were included in each bioassay in order to verify the susceptibility of the brine shrimps. LD₅₀ values were determined from 24 h counts, by Probit Analysis.³²

Acknowledgments

The authors acknowledge the financial support provided by FUNDECT - Fundação de Apoio ao Desenvolvimento do Ensino, Ciência e Tecnologia do Estado de Mato Grosso do Sul, Brazil. They are also thankful to CNPq for the fellowship granted. Thanks are also due to Dr. Norberto Peporine Lopes (Faculdade de Ciências Farmacêuticas de Ribeirão Preto/USP, SP, Brazil) for mass spectrometry analysis.

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Received: December 11, 2006

Web Release Date: December 13, 2007

Supplementary Information

Publication Dates

• Publication in this collection
  12 Feb 2008
• Date of issue
  2007

History

• Accepted
  13 Dec 2007
• Received
  11 Dec 2006