Recirculating chiller f 105

The leaves of Copaifera lucens Dwyer were collected from the field of the botanical garden of Rio de Janeiro, Arboreto, Canteiro, Brazil, identified by the botanist Haroldo Cavalcante de Lima, where a voucher sample was kept (RB 474303). The leaves were dried in a dark, dry and well-ventilated area. The leaves were air dried and ground using a knife mill, and 0.5 Kg of the powder was macerated three times each three days using 4 L of a hydroalcoholic solution (ethanol: water; 7:3), filtered and concentrated under vacuum (Rotavap: Model R210 Buchi ™ with Vacuum Pump V-700, Vacuum Controller V-855 and Recirculating Chiller F-105). The crude extract (88.58 g) was lyophilized. Fifty grams of the extract were dissolved in 600 mL of methanol: water (6:4), and partitioned with ethyl acetate (3 X 300 mL), followed by partition with n-butanol (4 X 200 mL), in sequence. The fractions were concentrated under vacuum and lyophilized. The yields were 14.4, 14.9 and 17.6 g for n-butanolic, ethyl acetate and water fractions, respectively. The n-butanolic fraction (BF) was analyzed by HPLC-UV and used in the biotransformation experiment. Gallic acid, as a reference substrate, was purchased commercially (Sigma-Aldrich).