Chemical Constituents of the Underground Stem Bark of Duguetia furfuracea ( Annonaceae )

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 β-N-óxido, de (-)-duguetina, dicentrinona, (-)-Nmetiltetraidropalmatina 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, α-gurjuneno, aromadendreno, biciclogermacreno, (E)-metil-isoeugenol e α-asarona foram isoladas a partir do óleo volátil, e as substâncias policarpol, óxido de β-cariofileno, 2,4,5-trimetóxi-estireno, α-asarona e asaraldeído foram obtidas do extrato em éter de petróleo. Este estudo descreve pela primeira vez o alcalóide β-N-óxido de (-)-duguetina, e a ocorrência das substâncias (-)-Nmetiltetraidropalmatina 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". 2In 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. 4On 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.
6][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 isoquinolinederived skeleton, 6 and the trypanocidal activity of some of these compounds.One of these papers 7 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.
The 1 H and 13 C 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.
The presence of 1,2-methylenedioxy and 9,10dimethoxy groups was indicated in the NMR spectrum  (9)  55.9 3.84 (s) OCH 3 (10)  55. 8  3.90 (s) 1 H and 13 C NMR spectra were acquired in CDCl 3 at 300 and 75 MHz respectively.TMS was used as internal standard, chemical shifts are shown in the δ scale with J values (Hz) in parentheses.
by two singlet signals at δ 3.84 (s) and 3.90 (s), and the typical pair of doublets of methylenedioxy protons was observed at δ 6.10 and 5.93 (d, 1.1 Hz).Also, were observed aliphatic protons signals at δ 3.59 (m) and 2.91 (m).These 1 H NMR data (Table 1) are in agreement with an aporphine alkaloid having 1,2methylenedioxy and 7-hydroxy groups with trans configuration at 6a,7, exhibiting a large coupling constant for the two doublets at δ 5.08 (d, 12.1 Hz) and 4.34 (d, 12.1 Hz).
The 13 C 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. 8By comparing the 13 C NMR data of 1 with those of 2, using N-O models available in the literature, namely oliveroline β-N-oxide (6a,7α-H, β-Noxide) 9 and dasymaschaline α-N-oxide (6a,7α-H, α-Noxide), 8 and confirming with NOESY the stereochemistry of the N-oxide moiety of 1, it was possible to propose that 1 is a (-)-duguetine β-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. 10This metabolite belongs to the ureide class and plays an important role in the transport and assimilation in some nitrogen-fixing species. 11Allantoin, 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. 10he 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. 13Essential 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 noteworthy feature is the fact that the isolation of aromatic compounds such as propenylbenzenes (or vinylbenzenes) has been described predominantly in the underground parts. 14,15Phenylpropanoid-derived compounds are widespread among vascular plants and play an important role in chemical defense. 16ith 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. 17he fresh volatile oil and the petroleum ether and alkaloid extracts were active in the brine shrimp lethality bioassay, with LD 50 values of 2.6, 6.1, and 36.9 μg mL -1 , respectively.

General experimental procedures
Optical rotations were measured on a Perkin-Elmer 341 polarimeter in MeOH.The FTIR spectra of either KBr pellets or CHCl 3 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 1 H and 13 C 1D and 2D NMR spectra were recorded at 300 MHz ( 1 H) and 75 MHz ( 13 C) 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 airdried and finely ground bark of the underground stem until pH 9.0 was reached and then subjected to exhaustive extraction with CHCl 3 .The resulting chloroform layer was concentrated under reduced pressure and a brown residue (68 g) was obtained.
The residue was solubilized in CHCl 3 and extracted with 5% HCl; the resulting acid fraction was adjusted to pH 9.0 (NH 4 OH) and again exhaustively extracted with CHCl 3 .
From the ethanol extract of the underground stem, the ureide allantoin (6) was isolated by precipitation with acetone.
The oil was fractionated by CC on silica gel (70-230 mesh), and eluted with petroleum ether:CHCl 3 :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 (160.2mg), 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 CHCl 3 providing 13 (176.4mg).The resulting extract (5 g) was then fractionated by CC on silica gel (70-230 mesh) using hexane:CHCl 3 :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)

Table 1 .
NMR spectroscopic data of compound 1