The first flavonoid isolated from Bromelia laciniosa ( Bromeliaceae )

1 Center for Studies and Research of Medicinal Plants, Federal University of San Francisco Valley, Petrolina, Pernambuco, Brazil. 2 Department of Chemistry of Natural Products, State University of North Fluminense, Campos dos Goytacazes, Rio de Janeiro, Brazil. 3 Museum of Botany, University of Bergen, Bergen, Norway. 4 Centre for Pharmacy, Department of Chemistry, University of Bergen, Bergen, Norway.


INTRODUCTION
The Bromeliaceae family has a predominantly neotropical distribution and includes about 58 genera and 3,200 species (Luther, 2010).The phytochemistry of this family is distinguished by the presence of triterpenoids and flavonoids.Other classes of compounds such as sterols, diterpenoids, cinnamic acid derivatives, substituted glycerols, lignans and nitrogen-containing compounds, among others, have also been identified in this family, although to a more limited extent.The distribution of flavonoids in leaves of species of Bromeliaceae was previously reported by Williams et al. (1978).Since the large number of species of Bromeliaceae, only few of them have been analyzed with respect to their chemical constituents so far (Manetti et al., 2009).
Several species of the genus Bromelia are being studied with respect to their chemical constituents and their pharmacologic activity.For example, Camacho-Hernandez et al. (2002)  The extracts of the leaves and branches of this species have also shown spasmodic activity, antiparasitic and cytotoxic activities (Payrol and Martinez, 2000;Raffauf et al., 1981;Geran et al., 1972).The biological potential of this species may be rationalized by the presence of diterpenes and flavonoids previously identified (Raffauf et al., 1981).Flavonoids also were identified in leaves of Bromelia karatas (Williams et al., 1978).Bromelia laciniosa is a species native to the Brazilian Caatinga and is known in the Northeast region of Brazil as "macambira" and "macambira de porco".The plant is used in the alimentation of man and domestic animals, especially in times of drought (Dutra et al., 2010).From the base of the leaves is extracted a mass, wherefrom a type of bread is produced (Angelim et al., 2007).The main therapeutic indications are for treatment of child colic, diarrhea, fever, jaundice, dandruff and hepatitis (Albuquerque et al., 2007).The decoctions of the roots is also popularly used against hepatitis and intestinal disorders and as a diuretic, while the dried and powdered leaves are used in cooking as a source of proteins (Agra et al., 2007).
In the present paper, we report results of the first phytochemical study of the species B. laciniosa and the isolation and chemical characterization of a flavonoid described for the first time in the Bromeliaceae family.

General experimental procedures
Mass spectra of the fraction were obtained on a gas chromatograph coupled to a mass spectrometer (QP2010 Plus, Shimadzu, Japan) with electron impact (EI) ionization, operating at an MS ionization voltage of 70 eV.The chromatograph was equipped with a fused silica capillary column FactorFour VF-5ms (30 m × 0.25 mm × 0.25 mm), and helium (He) was used as the carrier gas.The following chromatographic conditions were used: injector temperature at 250°C, detector temperature at 230°C; gas flow 1.0 ml/min; split 1/20; initial column temperature of 100°C with heating to 290°C at 3°C/min. 1 H and 13 C NMR spectra were recorded at 500.13 and 125.76 MHz for 1 H and 13 C, respectively, on a Bruker Avance DRX-500 instrument, using Methanol-D3 (Sigma Aldrich, USA) as solvent.The residual solvent signal ( 1 H) and the solvent signal ( 13 C) were used for spectral calibration.Chemical shifts were reported in units (ppm) and coupling constants (J) in Hz.Silica gel 60 (Merck, Kiesegel 60 F254) was used for analytical thin layered chromatography (TLC) and Silica gel 60 (Merck, 230 to 240 mesh) was used for column chromatography (CC).Spots on chromatograms were detected under UV light (254 and 365 nm).When necessary, vanillin sulfuric and NR + PEG reagents were used to detect the spots on the TLC plates.

Plant
The mature and healthy leaves of B. laciniosa Mart.ex Schult.f. (Bromeliaceae) were collected in the city of Petrolina (Coordinates 08° 59' 16.90" S and 40° 35' 20.60" W), State of Pernambuco, Brazil, in January, 2011.At the collection site, the plant was fully exposed to the sun.The samples were collected and identified by André Paviotti Fontana, a botanist from Centro de Recuperação de Áreas Degradadas da Caatinga (CRAD).A voucher specimen (6442) was deposited at the Herbário Vale do São Francisco (HVASF) of the Universidade Federal do Vale do São Francisco.

Extraction and isolation
The dried and pulverized leaves of B. laciniosa (879 g) were subjected to maceration with 95% EtOH for 72 h.The solution was filtered and concentrated under reduced pressure on a rotatory evaporator at 50°C, producing 39 g of crude ethanol extract (Bl-EtOH, 4.39%).For the phytochemical study, Bl-EtOH was suspended in MeOH:H2O (3:7 v/v) and partitioned with hexane, chloroform (CHCl3) and ethyl acetate (AcOEt) in increasing order of polarity to obtain the respective extracts.The chloroform fraction (Bl-CHCl3) was subjected to column chromatography over silica gel eluting with hexane, CHCl3, AcOEt and MeOH alone or in binary mixtures, in increasing order of polarity.In total, 113 fractions were collected (each 100 ml).The fractions were monitored by TLC and combined according to the similarity between the retention factors (Rf), resulting in different groups.The combined fractions 75 to 80 were purified by recrystallization using CHCl3 to afford the flavonoid 1 (14 mg).
The analysis of all spectral data for 1, including the 1D and 2D NMR spectra, led to the elucidation of its structure as the flavonoid 5,7-dihydroxy-3,3',4'trimethoxyflavone.The structure was corroborated by the mass spectral data.The EIMS revealed a molecular ion at m/z 344, consistent with the molecular formula C 18 H 16 O 7 (11 degrees of unsaturation) compatible with a substituted flavonol skeleton.In addition, two fragment ions were observed at m/z 165 and m/z 153, suggesting the fragmentation patterns for the A-ring and the B-ring, respectively.The observed fragmentations of 1 are rationalized in Figure 2 and confirm the NMR data.
It is noteworthy that until now phytochemical studies on B. laciniosa have been completely absent in current literature.Furthermore, the isolation of 5,7-dihydroxy-3,3',4'-trimethoxyflavone is being reported, to the best of our knowledge, for the first time in the Bromeliaceae family.In a leaf survey of 61 species of the Bromeliaceae, an unexpectedly wide spectrum of flavonoid constituents was encountered.The family is unique amongst the monocotyledons in the frequency and variety of flavonoids with extra hydroxylation or methoxylation at the 6-or 8-position.Quercetin (5-and 7hydroxy substituted flavone) was the most common leaf constituent and was identified as the 3-rutinoside or the 3-glucoside in some Bromeliaceae species (Williams et al., 1978).In a recent review about the secondary metabolites from Bromeliaceae family (Manetti et al., 2009), 25 flavonols were reported among 76 flavonoids identified in 83 species.Therefore, it may be considered that flavonols are widespread within the family but the occurrence of quercetin 3,3',4'-trimetyl ether was not reported.

Figure 1 .
Figure 1.Chemical structure of the flavonoid quercetin 3,3',4'-trimethyl ether (1) with highlight to A, B and C rings of the generic structure of flavonoids.

Figure 2 .
Figure 2. Main fragmentation patterns observed in the mass spectrum of 1.

Table 1 .
1 H (500 MHz) and 13 C (125 MHz) NMR data for 1 including results obtained by heteronuclear 2D shift-correlated HMQC and HMBC spectra, in MeOD as solvent and TMS as internal reference.Chemical shifts in δ (ppm) and coupling constants (J, in parenthesis) in Hz.