Development of an effect-directed analysis via high-performance thin-layer chromatography-bioassay-mass spectrometry (HPTLC-bioassay-MS) platform for identification of bioactive compounds in cherimoya fruit (Annona chirimola Mill.)

Abstract

Annona cherimola Mill. is a small tree cultivated in several countries including Chile. This tree produces a fruit called cherimoya, a good tasting fruit used in traditional medicine. Some beneficial biological activities for health have been demonstrated by in vitro assay, however few studies have identified the active compounds responsible for its bioactivity. The study of the bioactive components of cherimoya would allow the discovery of molecules with therapeutic potential for chronic non-communicable diseases, thus giving added value to the fruit and by-products generated. For the determination of bioactive compounds, an effect-directed HPTLC-bioassay-MS analytical platform was developed and implemented allowing the in situ identification of bioactive compounds through a simple and direct workflow, which allows a screening of the composition of peel, pulp and seed of cherimoya. The use of this analytical tool allowed the detection of three compounds with inhibitory activity on the acetylcholinesterase enzyme in the cherimoya peel, then they were studied by HPTLC-MS and UHPLC-DAD-MS/MS. The compounds were identified as anonaine, glaucine, and a third candidate as xilopine, the inhibitory capacity of these alkaloids was reported for the first time. Applying the same system, three phenolamides present in peel and seed were detected and identified as inhibitors of α-glucosidase (N-trans-feruloyl tyramine, N-trans-p-coumaroyl tyramine and N-trans-feruloyl phenethylamine). The first time this bioactivity is reported as well as the presence of the latter in cherimoya. The antioxidant compounds present in the peel and seed extract were detected via the HPTLC-DPPH assay, and the antioxidant capacity was quantified from the same assay by digital image processing using ImageJ software. The compounds with the highest activity, expressed as caffeic acid equivalents, were directly studied by TLC-MS analysis and preliminarily classified as acetogenins and phenolamides. In this way, the HPTLC-DPPH-MS assay was a complete methodology that allows to detect, quantify and identify antioxidant compounds, establishing the applicability of digital image processing in HPTLC-bioassay methodologies. The present work also determined a prominent inhibitory activity of AChE, α-glucosidase and antioxidant capacity of a cherimoya peel extract obtained through an optimized supercritical fluid extraction methodology. The bioactive compounds identified by the analytical platform were also quantified by UHPLC-ESI-MS analysis in the extracts. Additionally, leaves of different varieties of cherimoya were evaluated with the same methodology, establishing their potential as a source of AChE-inhibiting alkaloids. In summary, the application of the analytical platform made it possible to evaluate the potential of agroindustrial by-products, peel and seed, as a source of bioactive compounds with therapeutic potential.