Phytochemical Screening and Isolation of New Ent-Clerodane Diterpenoids from Croton guatemalensis Lotsy

Phytochemical screening of an ethanol–water extract (EWE) from the bark of Croton guatemalensis led to the isolation and identification of eight compounds, among them: five ent-clerodane diterpenoids [junceic acid (1), 6(s)-acetoxy-15,16-diepoxy-ent-cleroda-3,13(16),14-trien-20-oic acid (crotoguatenoic acid A) (2), 6(s)-hydroxyoxy-15,16-diepoxy-ent-cleroda-3,13(16),14-trien-20-oic acid (crotoguatenoic acid B) (3), formosin F (4), bartsiifolic acid (5)], and three flavonoids [rutin (6), epicatechin (7), and quercetin (8)]. Of these, 2 and 3 are reported here for the first time. Structures were established through conventional spectroscopy methods and their absolute configurations were determined by optical rotation and comparison of experimental electronic circular dichroism (ECD) and theoretical calculated ECD spectra. A suitable high performance liquid chromatography (HPLC) method for quantifying rutin (6) was developed and validated according to standard protocols. Affinity-directed fractionation was used to identify possible in vitro active compounds on α-glucosidases from Saccharomyces cerevisiae. HPLC-ESI-MS was used to identify the inhibitors as free ligands after being released from the enzymatic complex by denaturing acidic conditions. The affinity studies led to the identification of ent-clerodane diterpenoids as active compounds. In silico analysis allowed us to determine the best conformational rearrangement for the α-glucosidase inhibitors.