New Antioxidant Caffeate Esters of Fatty Alcohols Identified in Robinia pseudoacacia

The stem bark of black locust (Robinia pseudoacacia L.) was extracted, and nine antioxidant compounds (R1-R9) were detected by high-performance thin-layer chromatography combined with the radical scavenging 2,2-diphenyl-1-picrylhydrazyl (DPPH•) assay, multi-detection, and heated electrospray high-resolution mass spectrometry. For structure elucidation, the methanolic crude extract was fractionated by solid-phase extraction, and the compounds were isolated by reversed-phase high-performance liquid chromatography with diode array detection. The structures of isolated compounds were elucidated by nuclear magnetic resonance and attenuated total reflectance Fourier-transform infrared spectroscopy as well as gas chromatography-mass spectrometry to determine the double bond position. 3-O-Caffeoyl oleanolic acid (R1), oleyl (R2), octadecyl (R3), gadoleyl (R4), eicosanyl (R5), (Z)-9-docosenyl (R6), docosyl (R7), tetracosyl (R8), and hexacosanyl (R9) caffeates were identified. While R1 has been reported in R. pseudoacacia stem bark, the known R3, R5, R7, R8, and R9 are described for the first time in this species, and the R2, R4, and R6 are new natural compounds. All nine caffeates demonstrated antioxidant activity. The antioxidant effects of the isolated compounds R1-R8 were quantified by a microplate DPPH• assay, with values ranging from 0.29 to 1.20 mol of caffeic acid equivalents per mole of isolate.