Punica granatum L. Leaf Extract Attenuates Lung Inflammation in Mice with Acute Lung Injury

Pinheiro, Aruanã Joaquim Matheus Costa Rodrigues; Gonçalves, Jaciara Sá; Dourado, Ádylla Wilenna Alves; de Sousa, Eduardo Martins; Brito, Natilene Mesquita; Silva, Lanna Karinny; Batista, Marisa Cristina Aranha; de Sá, Joicy Cortez; Monteiro, Cinara Regina Aragão Vieira; Fernandes, Elizabeth Soares; Monteiro-Neto, Valério; Campbell, Lee Ann; Zago, Patrícia Maria Wiziack; Lima-Neto, Lidio Gonçalves

The chemical constituents of EAFPg were analyzed in a high-performance liquid chromatography (HPLC) system (LC-10AD, Shimadzu) equipped with a photodiode array detector coupled to an Esquire 3000 Plus ion trap mass spectrometer (Bruker Daltonics, Bremen, Germany), using electrospray ionization (ESI) as previously described [10] . Separation was performed using a Phenomenex Kinetex C-18 column (250 × 4.6 mm, 5 μm; Torrance, CA, USA). The column oven was maintained at room temperature. The HPLC was set up with an elution gradient as follows: 0-2 min, 5% B; 2-10 min, 5-25% B; 10-20 min, 25-40% B; 20-30 min, 40-50% B; 30-40 min, 50-60% B; and 40-50 min, 70-100% B. Acetic acid (2%) in Milli-Q water was used as mobile phase A, and methanol was used as mobile phase B. The injection volume consisted of 25 μl of the reconstituted sample at 5 mg/ml, with a flow rate of 0.6 ml/min. Detection was achieved in a diode array detector (DAD) at 200-500 nm and with direct mass spectrometry/mass spectrometry, a method in negative electrospray (-ESI) mode with a detector voltage maintained at 4.0 kV, ion source at 40 V, and capillary temperature at 320°C. The nebulizing gas was nitrogen (N 2 ) flowing at 7 ml/min, a sheath gas provided at a pressure of 27 psi, while helium was used as the collision gas. Analyses were carried out using full-scan mass spectra and data-dependent MS 2 scans from m/z 100 to 2000. The compounds were identified on the basis of their molecular ion mass fragmentation. The obtained mass spectrum was compared with that of the literature.