A New Flavonoid Derivative from Leaves of Oxandra sessiliflora R

A fração em acetato de etila (EtOAc) obtida a partir da partição do extrato de etanol (EtOH) das folhas de O. sessiliflora R. E. Fries (Annonaceae) foi submetida a diversos procedimentos cromatográficos, incluindo cromatografia líquida de alta eficiência (HPLC), o que resultou no isolamento dos flavonóides: quercetina-3-O-α-L-ramnopiranosil-(1→4)-β-D-glucopiranosídeo (1), inédito na literatura, canferol-3-O-α-L-ramnopiranosil-(1→4)-β-D-glucopiranosídeo (2), rutina (3) e canferol-3-O-rutinosídeo (4). As estruturas foram definidas através da análise dos espectros de ressonância magnética nuclear (NMR) de H e de C (1D e 2D) e espectrometria de massas.


Introduction
The genus Oxandra (Annonaceae) consists of about 22 species, 14 of these being found in Brazil and distributed in North, Northeast, Midwest and Southeast regions. 1,2his genus has native origin and phytogeographic domains in the Amazon, Caatinga, Cerrado, and Atlantic Forest. 3There are few articles reporting the chemical composition and pharmacological activity of plants of the genus Oxandra.Alkaloids, triterpenes, monoterpenes, and steroids with anti-inflammatory and antioxidant activities were isolated from O. xylopioides, [4][5][6][7][8][9][10][11][12] while trypanocidal and antileishmanial monoterpenes have been reported from O. espintana. 13Additionally, alkaloids, sesquiterpenes and triterpenes have been isolated from O. asbeckii. 14andra sessiliflora R. E. Fries, popularly known as "conduru-preto", 3,15,16 is a species endemic to Brazil in which only the chemical composition of essential oil from leaves have previously been reported in the literature. 17n continuation with our studies on O. sessiliflora, the present work describes the isolation and characterization of four flavonoids from ethyl acetate (EtOAc) phase from ethanol (EtOH) extract from leaves: quercetin- The structures of the isolated compounds were established based on the analysis of their 1 H and 13 C nuclear magnetic resonance (NMR) spectra, including HMQC, HMBC and COSY experiments, and comparison with literature data.This is the first occurrence of flavonoid 1 and assignment of 13 C NMR data of flavonoid 2.

Results and Discussion
The EtOH extract of the leaves of O. sessiliflora was partitioned between MeOH:H 2 O 2:1 and hexane, CH 2 Cl 2 and EtOAc successively.The EtOAc fraction was subjected to column chromatography on reverse phase (C 18 ) and Sephadex LH-20, followed by purification of the obtained fractions by high performance liquid chromatography (HPLC) to afford compounds 1-4 (Figure 1).
The 1 H NMR spectrum of 2 showed similarities to that recorded to flavonoid 1, with two broad singlets at d H 6.19/6.38,assigned to H-6/H-8 of ring A. This spectrum displayed also signals at range from d H 3.20 to 5.21 (oxymethine hydrogens) and one doublet at d H 1.24 (J = 6.0 Hz), suggesting the presence of rhamnose in the molecule.The signals superimposed at d H 5.21 (2H) have been assigned to the anomeric protons H-1" and H-1'''.The main observed difference in the 1 H NMR spectrum of 2 is associated to the substitution pattern of kaempferol (1,4-disubstituted B ring), due to the presence of two doublets at d H 6.88 and 8.03 (d, J = 8.0 Hz) integrated to two hydrogens each and thus assigned to H-3'/H-5' and H-2'/H-6', respectively. 13  and 102.7 (C-1'"), confirming the presence of glucose and rhamnose.These information, associated with literature data for flavonoids with the same aglycone, 19 allowed the identification of 2 as kaempferol-3-O-β-D-glucopyranosyl-(1→4)-α-L-rhamnopyranoside, previously isolated from Acacia pennata Willd (Mimosaceae). 20However, this is the first occurrence in Annonaceae family and the first description of its assigned 13 C NMR data.
The structures of flavonoids 3 and 4 were identified by analysis of 1 H and 13 C NMR as well as HRESIMS and comparison with data described in the literature. 18,19

Experimental
General procedures 1 H and 13 C NMR spectra were obtained on Varian spectrometer-model INOVA, operating at 500 MHz for 1 H and 125 MHz for 13 C using CD 3 OD as a solvent and tetramethylsilane (TMS) as internal reference.HRESIMS spectrum (negative mode) was recorded on a Bruker Daltonics UltrOTOFq-ESI-TOF spectrometer.Silica gel (70-230 mesh, Merck) and Sephadex LH-20 (Amersham Biosciences) were used for column chromatography (CC), whereas silica gel 60 GF 254 was employed for analytical

Plant material
The leaves of O. sessiliflora were collected in the Environmental Park of Teresina-PI, in June 2009.The species was identified by Professor Roseli Farias Melo Barros and a voucher specimen with number TEPB 27870 was deposited in the Herbarium Graziela Barroso do Amaral (UFPI).

Extraction and isolation
The leaves of O. sessiliflora were dried at room temperature and then grinded.The obtained material (779 g) was subjected to exhaustive maceration with EtOH at room temperature.After concentration on reduced pressure, 109 g of EtOH extract were obtained (14%).Part of the EtOH extract (86 g) was suspended in MeOH-H 2 O (2:1) and extracted with hexane, CH 2 Cl 2 and EtOAc successively to afford 21 g (24%), 30 g (35%) and 14 g (17%) of organic phases, respectively.

Figure 2 .
Figure 2. HMBC and COSY correlations in the structure of 1.

Table 1 .
NMR data of compounds 1 and 2 (500 MHz and 125 MHz, d in ppm, J in Hz, CD 3 OD)