Flavonoids from Roots of Dahlstedtia glaziovii ( Fabaceae )

O estudo fitoquímico das raízes de Dahlstedtia glaziovii (Fabaceae) forneceu um novo dibenzoilmetano (glaziovione), juntamente com dezoito compostos conhecidos. Suas estruturas foram determinadas através de métodos espectroscópicos de ressonância magnética nuclear (NMR) 1D e 2D (correlação heteronuclear única quântica, HSQC, e correlação heteronuclear de múltipla ligação, HMBC) e espectrometria de massas de alta resolução (HRMS). A atividade antiproliferativa foi investigada para os extratos, para os dibenzoilmetanos 2’-metóxi-8-(α-α-dimetilalil)furano-[4”,5”:3’,4’]-dibenzoilmetano, 3,4-metilenodioxi-2’-metóxi-8-(α-α-dimetilalil)-furano[4”,5”:3’,4’]-dibenzoilmetano e pongamol, e para as flavonas lanceolatina B, karanjina, pongapina e 3’,4’-metilenodioxi-2’’,2’’-dimetilpirano-[5’’,6’’:8,7]-flavona. Os dibenzoilmetanos foram mais ativos do que as flavonas. A atividade antimicrobiana foi investigada para os extratos, sendo que estes não se apresentaram ativos.


Introduction
The genera Dahlstedtia Malme and Lonchocarpus Kunth (Fabaceae) are very similar in terms of taxonomy as well as in the biosynthesis of secondary metabolites.A recent study investigated the relationships of Lonchocarpus and allied genera based on nuclear and plastid DNA markers. 1This genus is native to the southern portion of South America, including southern and southeastern Brazil.Dahlstedtia glaziovii (Taub.)M.J. Silva & A.M.G.Azevedo (previously Lonchocarpus glaziovii Taub.) is restricted to Rio de Janeiro state, municipalities of Nova Friburgo and Alto Macaé, where it is popularly known as "Guaraná-timbó". 1 Dahlstedtia has been characterized by producing prenylated flavonoids, usually containing furan and dimethylpyran moieties. 2lavonoids are one of the largest groups of secondary metabolites, with a wide range of biological activities including antioxidant, 3 photoprotective, 4,5 antimicrobial, 6 anti-inflammatory, 7 anticancer, 8,9 memory effects and reduction of cardiovascular diseases, 10 antifungal, 9,11 and antiviral, 9 among others. 12n this context and continuing our research on native Fabaceae species, we conducted a phytochemical investigation of the roots of D. glaziovii, which led to the isolation and structural determination of a new dibenzoylmethane (1), in addition to eighteen known flavonoids (2-19) (Figure 1).Antibacterial and anti-Candida activities were assessed for the crude extracts.The antiproliferative in vitro activity of the extracts and some flavonoids was evaluated.

General experimental procedures
Fourier transform infrared (FTIR) spectrum was acquired using a BIORAD FTS-3500 FTIR spectrometer, in CHCl 3 and as KBr discs.The UV-Vis spectra were obtained in CHCl 3 on a UV-2401PC (Shimadzu) spectrophotometer system.Optical rotations were measured in CHCl 3 solutions at room temperature on a Jasco polarimeter model P-2000.High-resolution mass spectra were run on a quadrupole LC-MS MicroQTOF II (Bruker) spectrometer equipped with an electrospray ionization source.Nuclear magnetic resonance (NMR) data were recorded at ambient temperature in CDCl 3 on a Bruker Avance 400, operating at 9.4 T, observing 1 H and 13 C at 400.1 and 100.6 MHz, respectively, and on a Bruker Avance III 600 MHz, operating at 14.1 T ( 1 H at 600 MHz; 13 C at 150 MHz) equipped with 5 mm cryo probe with automatic tuning matching (ATMA) and field gradient in z.The chemical shifts (d) are given in ppm relative to tetramethylsilane (TMS, d 0.00 ppm) as internal standard and coupling constants (J) in Hz.High-performance liquid chromatography (HPLC) analyses were performed on a Waters chromatograph consisting of quaternary pump, auto injector, 2998 PDA detector and coupled to an Empower software data acquisition system.The analyses were carried out using an analytic reversed phase column X-Terra C18, 5 μm, 250 × 4.6 mm and guard column at 20 ºC (Waters).The mobile phase consisted of an isocratic elution from acetonitrile:water (55:45, v/v), flow rate of 1 mL min -1 .The acetonitrile (ACN) was HPLC grade, filtered under vacuum through nylon membrane (0.45 μm) of Millipore ® and degassed for 30 min in ultrasound bath.Ultrapure water was obtained by Milli-Q system.The samples were filtered through Millex ® HV PVDF (0.45 μm) of Millipore ® (Cork, Ireland).The separations of fractions by HPLC were carried out using a semi-preparative column X-Terra C18, 10 μm, 300 × 7.8 mm (Waters).For open column chromatography silica gel 60 (70-230 mesh) was used whereas in flash column chromatography silica gel (230-400 mesh, Merck) was used.Radial chromatography was performed on Chromatotron 7924T (Harrison Research Chromatotron, San Bruno, California, USA) using silica gel (60 GF 254 Merck, 1 mm).Aluminum pre-coated silica-gel plates (60 F 254 Merck, 0.25 mm) were used for thin layer chromatography (TLC) analyses and glass pre-coated silica-gel plates (60 PF 254 Merck, 1 mm) were used for preparative TLC.The spots were detected by spraying with p-anisaldehyde reagent, followed by heating.

Plant material
The roots of Dahlstedtia glaziovii were collected in August 2009, in Nova Friburgo (Rio de Janeiro state, Brazil) at 22º20'2.2" S, 42º41'42.3"W and 1.221 m high, and identified by the taxonomists Prof Ana Maria Tozzi from Unicamp and Prof Marcos Silva from UFG.A voucher specimen (Marcos J. Silva 1077) was deposited at the Herbarium UEC, Institute of Biology, Unicamp.
Considering the mean value of GI 50 , the PE and CH 2 Cl 2 extracts showed better antiproliferative activity, with mean GI 50 of 5.1 and 7.1 μg mL -1 , respectively.The MeOH extract showed weak activity, with a mean GI 50 of 57.4 μg mL -1 .Both PE and CH 2 Cl 2 extracts showed a similar activity against the cell lines U251 (glioma), MCF7 (breast) and 786-0 (renal); the PE extract inhibited the HT29 (colon) cell line more strongly than the CH 2 Cl 2 extract.Although they showed similar results for antiproliferative activity, the CH 2 Cl 2 extract was obtained in a larger amount than the PE extract.Therefore, the CH 2 Cl 2 extract was fractionated and some of the isolated flavonoids (2-4, 11-13 and 18) were isolated in sufficient quantity to assay against tumor cell lines.
These results suggest that the cyclization to form the C ring decreased the antiproliferative activity, since the flavonoids with an open chain (the central part of the molecule) were more active (dibenzoylmethanes 2-4) than those with a C ring (flavones 11-13 and 18).
Comparison of dibenzoylmethanes 2 and 3 indicated that the presence of the methylenedioxy group in 3 decreased the mean GI 50 activity (25.1 and 32.0 μmol L -1 , respectively).Comparing compounds 2 and 4, the structural difference is the presence of the α,α-dimethylallyl group at C-8 in 2, which leads to an increase of the mean GI 50 activity (25.1 and 33.7 μmol L -1 , respectively).
Evaluating the antiproliferative activity on the nontumor cell line HaCat (keratinocyte), the extracts and the prenylated flavonoids affected the proliferation of these cells within the same order of magnitude as their effect on tumor cells.The same trend was observed for the chemotherapeutic agent doxorubicin, while the prenylated flavonoids assayed showed less toxicity than doxorubicin.Thus, in vivo investigation is needed to determine the safety of using these flavonoids as potential chemotherapeutic agents.

Conclusions
The isolation of the prenylated flavonoids (1-19) from D. glaziovii roots concurs with the reclassification suggested by da Silva et al.. 1 Flavonoids with prenyl cyclized groups such as a furan ring and 2",2"-dimethylpyran, which have been used as indicators of the evolution of the genus Dahlstedtia, are found at the angular position in the main skeleton. 2any compounds isolated from D. glaziovii have been found in other species, including D. pinnata 2 (Benth.)Malme and D. penthaphylla 2 (Taub.)Burkart.This indicates the chemical similarity between the species, in concordance with the morphological affinity cited by da Silva et al.. 1 In addition, other species reclassified as Dahlstedtia, 1 previously studied, have some chemical similarity to  and D. muelbergiana (Hassl.)M.J. Silva & A.M.G.Azevedo (L.muehbergianus Hassl.). 29ibenzoylmethane 1 is a new metabolite, and flavonoids 6, 7, 13 and 19 are reported for the first time in the genus Dahlstedtia.This is the second report of dibenzoylmethane 6, which was isolated for the first time in Muellera montana M.J. Silva & A.M.G.Azevedo (Lonchocarpus montanus). 20he antiproliferative assays showed that the members of the dibenzoylmethane series were more active than the flavones, and also showed that these flavonoids have promising activity.

Table 1 .
1H and 13 C NMR spectral data a for compounds 1 and 2 a