Chemical constituents of Distictella elongata ( Vahl ) Urb . ( Bignoniaceae )

Pectolinarin, a flavone heteroside, was isolated from Distictella elongata (Vahl) Urb. leaves ethanol extract, along with a mixture of ursolic, pomolic and oleanolic acids, besides β-sitosterol. Their structures were established on the basis of spectral analysis (1H and 13C NMR, 1D and 2D) and they were compared with literature. This is the first report on the occurrence of this flavonoid in a species of the Bignoniaceae family.


INTRODUCTION
Bignonieae is a large and diverse clade of neotropical lianas.The group is widely distributed in the neotropics, occurring in Central America, Amazonia, the Atlantic forests of eastern Brazil, and the open dry forests and savannahs of Argentina, Bolivia, Brazil, and Paraguay.Bignonieae contains all the lianas of the Brazilian Bignoniaceae and most of the species (approximately 250 of the 350) are in the four large genera, Arrabidaea, Adenocalymma, Anemopaegma and Memora (Lohmann 2006).Distictella Kuntze is a genus of 18 species in this tribe.The species are lianas or, less frequently, shrubs (Pool 2009).Distictella elongata (Vahl) Urb.appears as Distictis elongata Bureau & K. Schum. in the FLORA BRASILIENSIS and its actual name is Amphilophium elongatum (Vahl) L.G. Lohmann (Cipriani et al. 2007, Bedir et al. 2009, Lohmann 2010).
Bignoniaceae is characterized by the presence of terpenoids, flavonoids, alkaloids, and special aromatic derivatives of the shikimic acid pathway (Cipriani et al. 2007).A β-lapachone derivative was also previously obtained from the roots of D. elongata (Bedir et al. 2009).
In the present study, leaves ethanol extract (LEE) from D. elongata were phytochemically investigated affording the flavonoid di-O-glycoside pectolinarin (1) along with a mixture of the triterpenoids: ursolic, pomolic and oleanolic acids and β-sitosterol.

GENERAL EXPERIMENTAL PROCEduRES
Optical rotation was measured on a Bellingham + Stanley Ltda ADP 220 polarimeter, whereas Infrared (IR) spectra were recorded on a Perkin-Elmer Spectrum One spectrophotometer.Melting 873-879 LEANDRO R. SIMÕES et al.
point was determined on an electrothermal digital apparatus (model MQAPF-30; Microquímica, Brazil), without correction.UV spectra were measured in a UV-2900 UV-VIS recording spectrophotometer (HITACHI, Japan).NMR spectra were obtained in dMSO-d6 with TMS as internal standard and they were recorded on a Bruker Avance DRX-400 equipment, (DQ/ICEX/ UFMG).The MS system used was a quadrupole time-of-flight instrument (ultrOTOF-Q, Bruker Daltonics, Billerica, MA, U.S.A) equipped with an ESI positive and negative ion source.The analyses were performed with the mass spectrometer in positive mode.The following settings were applied throughout the analyses: capillary voltage 4500 V; dry gas temperature 150 °C; dry gas flow 4 L/min.; nebulizer gas nitrogen.(Castilho and Kaplan 2008).The n-hexane-EtOAc 7:3 fraction 51-73 (99 mg) was submitted to preparative TLC using CH 2 Cl 2 -EtOAc (3:2) as eluent affording a mixture (17 mg) of ursolic, pomolic and oleanolic acids that were identified by 1 H and 13 C NMR.A portion of the EtOAc:MeOh (1:1) fraction (230 mg) was further purified by preparative reverse phase hPLC separation to give the flavone pectolinarin (17 mg).
Pectolinarin Pectolinarin was purified on a Shimadzu hPLC system (Japan) composed of pump LC-8A, UV-Vis detector SPD-GAV, controller system SCL-8A and integrator C-R4A.An OdS column (250 × 20 mm I.D., 10 mm; Shimadzu, Japan) was employed at room temperature, at a flow rate of 4.0 mL/min and UV220 detection.The mobile phase was consisted

RESULTS AND DISCUSSION
The RP-HPLC profile of the LEE from D. elongata has shown the major peaks in the range of 19.0 to 31.0 min.The UV spectra registered online are characteristic of cinnamoyl derivatives: peaks with Rt 19.6 to 20.5 min; and flavones: peaks with Rt 26.5 to 31.0 min (Mabry et al. 1970).TLC phytochemical screening carried out for LEE indicated the presence of phenols, including flavonoids, terpenoids and/ or steroids (Wagner et al. 1984).
Fractionation of LEE by column chromatography (CC) on silica gel yielded eight fractions.TLC analysis of the EtOAc fraction has shown mainly the presence of terpenoids and/or steroids.It was further chromatographed over a flash silica gel chromatography column to give 17 mg of a mixture of triterpenoids characterized by 1 H and 13 C NMR spectroscopy.
The 1 H NMR spectrum showed many signals at region δ 0.66-2.00ppm corresponding to methyl and methylene hydrogens.The signal at δ 5.13 was attributed to the olefin hydrogen bonded to C-12 of ursane/oleanane triterpenoids (Castilho and Kaplan 2008). 13C NMR spectra have shown the chemical shift of the carbinol and olefin carbons that led to the identification of the three terpenoids: ursolic, pomolic and oleanolic acids (Mahato and Kundu 1994).LEANDRO R. SIMÕES et al.
TLC analysis of the EtOAc:MeOh (1:1) fraction has shown that it contained phenols only (Wagner et al. 1984).RP-HPLC-DAD analyses of the EtOAc and EtOAc:Meoh (1:1) fractions have shown a peak with retention time of approximately 31.0 min.The substance corresponding to this peak was isolated from the fraction EtOAc:MeOh (1:1), by preparative RP-hPLC, and was identified by spectrometric analyses.
The UV spectrum of the isolated substance was typical of a flavone ( λmax 273.5 and 328.5 nm).The presence of a free hydroxy group at C-5 and the absence of free hydroxy at C-4' and C-7 was indicated by the effects of AlCl 3 and NaOAc in the UV curve.The difference in band I in the methanol spectra (λ 328.5 nm) and after addition of NaOMe (λ 370.5 nm) is 42 nm with a decrease in intensity, indicating the absence of free Oh at C-7 and C-4'.No modification of the uv curve, relatively to the AlCl 3 curve, after addition of HCl, indicated the presence of a free 5-hydroxyl group (Mabry et al. 1970, Markham 1982).
The 1D 1 H NMR spectrum showed an AA'XX' system of spins at δ 8.04 and 7.17 ppm (2h each, J = 8.0 hz, 2' and 6'; 3' and 5', respectively) due to hydrogens in a 4′-oxygenated B ring.The doublets at 4.57 ppm (J = 1.1 hz, h-1'") and δ 5.12 ppm (J = 4.00 Hz, H-1") are typical of anomeric hydrogens.The singlet at δ 12.96 ppm for the hydroxy proton indicate the formation of a hydrogen bond with the neighboring oxygen.H-8 and H-3 appeared as singlets at δ 6.94 and 6.93 ppm, respectively.The doublet at δ 1.06 (3h, J = 4.0 hz, h-6'") was related to a rhamnose methyl group.Two methoxy singlets at δ 3.78 (4'-OCh 3 ) and 3.97 ppm (6-OCh 3 ) were observed and they were confirmed by the 1d 13 C NMR signals at δ 55.6 (4'-OCh 3 ) and δ 60.3 ppm (6-OCh 3 ).Nine signals were observed in the 13 C NMR in the range of δ 66.0-76.5 ppm, corresponding to two sugar units: D-glucose and L-rhamnose as confirmed by 2d hMBC and hSQC-TOCSY experiments.The methyl group of rhamnose gave a signal at δ 17.8 ppm in the 13 C spectrum.Acid hydrolysis of the isolated substance yielded an aglycone, pectolinarigenin, besides D-glucose and L-rhamnose which were confirmed by TLC.According to the coupling constant observed in the 1 h NMR spectrum, the configurations at the anomeric carbons of D-glucose and L-rhamnose were determined as β-(J = 1.1 hz) and α-linkages (J = 4.00 hz), respectively.
The chemical shifts of the carbon signals for D-glucose and L-rhamnose, indicated L-rhamnose as the terminal sugar.The C-6 glucose signal at δ 66.0 ppm indicated that the disaccharide should be rutinose (Moccelini et al. 2009).The DEPT experiment confirmed this signal for a methylene group.The linkage of the rutinosyl moiety to the oxygen atom attached to C-7 was confirmed by the hMBC correlation between the glucosyl h-1 (δ 5.12) and C-7 (δ 157.3).The 13 C NMR data also supported the attachment of the rutinosyl moiety to the oxygen at the 7-position of a flavone (Table I and Figure 1).
The antioxidant potential of seven Korean thistles rich in pectolinarin was evaluated via the peroxynitrite.The DPPH free radical assays exhibiting strong activity (Jeong et al. 2008).Pectolinarin isolated of the leaves of Cirsium setidens (Compositae), demonstrated hepatoprotective efficacy in a rat model of hepatic injury caused by D-galactosamine.It was suggested that the activity occurs mainly via SOd (superoxide dismutase) antioxidant mechanism (Yoo et al. 2008).
However, in the present study, pectolinarin has not presented any radical scavenging activity on the DPPH assay, although the LEE and the EtOAc:MeOh (1:1) fraction presented antioxidant activity by the DPPH assay (data not show).These results might be indicative of the presence of other phenols in LEE and EtOAc:MeOh (1:1) fraction which would be responsible for their antioxidant activity.
In vivo studies have demonstrated that oral administration of pectolinarin and a fraction rich in pectolinarigenin isolated from aerial parts of Cirsium chanroenicum at 20-100 mg/kg in several animal models resulted in inhibitory activities of inflammation/allergy: arachidonic acid-induced mouse ear edema, carrageenan-induced mouse  and C-6 (down field at δ 133.2 ppm) (Figure 1) (Silverstein et al. 2007, Yim et al. 2003).
The ESI-MS spectrum showed an ion peak at m/z 623 [M + H] + and ion peaks at 645 [M + Na] + and 661 [M + K] + , which are coherent with the molecular formula C 29 H 34 O 15 for the flavone, pectolinarin.
M: Multiplicity, J: coupling constant *Approximate shifts obtained by HSQC experiment.