Bio-guided optimization of the ultrasound-assisted extraction of compounds from Annona glabra L. leaves using the etiolated wheat coleoptile bioassay.

A bio-guided optimization of the extraction of bioactive components from Annona glabra leaves has been developed using the etiolated wheat coleoptile bioassay as the control method. The optimization of an ultrasound-assisted extraction of bioactive compounds using allelopathy results as target values has been carried out for the first time. A two-level fractional factorial experimental design was applied to optimize the ultrasound-assisted extraction. The solvent was the extraction variable that had the most marked effect on the resulting bioactivity of the extracts in the etiolated wheat coleoptile bioassay. Extraction time, extraction temperature and the size of the ultrasonic probe also influenced the bioactivity of the extracts. A larger scale extraction was carried out in the next step in the allelopathic study, i.e., the isolation of compounds from the bioactive extract and chemical characterization by spectroscopic techniques, including NMR. Eight compounds were isolated and identified from the active extracts, namely two steroids (β-sistosterol and stigmasterol), five diterpenes with the kaurane skeleton (ent-kaur-16-en-19-oic acid, ent-19-methoxy-19-oxokauran-17-oic acid, annoglabasin B, ent-17-hydroxykaur-15-en-19-oic acid and ent-15β,16β-epoxy-17-hydroxy-kauran-19-oic acid) and the acetogenin asimicin. The most active compound was annoglabasin B, which showed inhibition with values of -95% at 10(-3) M, -87% at 5×10(-4) M and greater than -70% at 10(-4) M in the etiolated wheat coleoptile bioassay.