Chemical Characterization of Alcoholic Crude Extract of Bauhinia Longifolia
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Chemical characterization of alcoholic crude extract of Bauhinia Longifolia using Time-Slice Liquid Chromatography–Solid-Phase Extraction–Nuclear Magnetic Resonance Aquino, A.J*; Alves, T.C; Ferreira, A. G; Cass, Q.B. Chemistry Department, Federal University of São Carlos, Brazil. (*[email protected]) Bauhinia (Fabaceae) genus are found in tropical regions of Asia, Africa, and Central and South America. In Brazil, these plants are known as ‘pata-de-vaca’ (cow’s foot) because of their leaf shape1. Tea made from the leaves of B. longifolia and B. forficata is consumed in the Brazil2 for its anti-diabetic properties. Although the widespread use, only recently that phytochemical investigations have revealed its chemical constituents. The phytochemical literature for longifolia species is, however, scarce being known only five well known flavonoids: guaijaverin, quercetin, quercitrin, isoquercetrin and hyperin3,4,5. NMR spectroscopy is a structural elucidation technique widely used in natural products because it can provide unequivocal information on chemical structures that can be complemented by mass spectrometry. The procedure of isolation and purification of compounds, however, takes a lot of time and effort. In this sense, the coupling of the LC to the NMR greatly accelerates this process, especially when using an automated SPE interface, which solves many incompatibilities between the systems. The trap in SPE cartridges allows the concentration of each chromatographic peak and also avoids the need of deuterated solvents for chromatographic separation. SPE trapping is usually carried out by ultraviolet absorptivity; however, trapping by retention time trapping is advantageous since it precludes the need of UV absorption. To meet this end, the chromatographic separation must have high reproducibility to not cause co-elution at the SPE trap. Using this latter approach with Restek Raptor Biphenyl ® (5 μm, 15 x 0.46 cm) column a 1 µL alcoholic crude extract (77 mg.ml-1) was injected at gradient elution (5-80% B; 60 min) with a flow rate of 1 mL.min-1 with the following mobile phase: A – water (with 100 uM formic acid) and B – methanol (with 100 uM formic acid). For column clean up 100% B was maintained for 10 min previously to re-equilibration a further 10 min at 5% B. Deuterated methanol (99,8% D) was used for compounds extraction from the SPE cartridges to the NMR tubes (Bruker, 3 mm o.d.). The NMR spectra were registered at 600MHz spectrometer. At these conditions, 20 chromatographic peaks were trapped and, so far, 4 compounds were unambiguously assigned from which 3 unknown to this species: afzelin, myrecitrin and trifolin. To add to the chemical characterization the chromatographic condition were reproduced using a LC-QqTof (with splitting of 330 µL.min-1 of the mobile phase to the ESI source). The complete chemical characterization will be fully discussed References 1. Filho, V. Chemical composition and biological potential of plants from the genus Bauhinia. Phytotherapy Research, v. 23, n. 10, p. 1347-1354, 2009. 2. Almeida, E. et al. Hypoglycemic effect of Bauhinia cheilandra in rats. Fitoterapia, v. 77, n. 4, p. 276-278, 2006. 3. dos Santos, A. et al. Quercetin and quercetin 3-O-glycosides from Bauhinia longifolia (Bong.) Steud. show anti-Mayaro virus activity. Parasites & Vectors, v. 7, n. 1, p. 130, 2014. 4. Duarte-Almeida, J.; Negri, G.; Salatino, A. Volatile oils in leaves of Bauhinia (Fabaceae Caesalpinioideae). Biochemical Systematics and Ecology, v. 32, n. 8, p. 747-753, 2004. 5. Salatino, A. et al. Foliar flavonoids of nine species of Bauhinia. Revista Brasileira de Botânica, v. 22, n. 1, p. 17-20, 1999. .