Larissa Carvalho Bezerra 1, André Assunção Ferreira2, Fábio Domingues Nasario3, Guilherme J. Zocolo4, João H. M. S. Sena5, Gisele Silvestre da Silva5, Marisa Jadna Silva Frederico 6, Caio Bruno Rodrigues Martins 1,2
Anti-α-Amylase Biomarkers from Spondiastuberosa Leaves: An Integrated In Vitro Assay, Biochemometric, and Molecular Docking Approach
Glycemic control in type 2 diabetes (T2D) can be effectively achieved by inhibiting α-amylase (α-AMY), a key enzyme involved in carbohydrate digestion. Natural inhibitors derived from plants have gained increasing attention due to their chemical diversity and lower toxicity compared to synthetic drugs. Spondias tuberosa, a species native to Brazil’s semi-arid region, presents a rich and diverse phytochemical profile with promising antidiabeticpotential, yet remains poorly explored at the molecular level.
In this study, we established and applied an integrated screening pipeline combining in vitro enzymatic assays, biochemometric analysis, and molecular docking to identify natural α-AMY inhibitors in S. tuberosa leaf extracts. Two extraction techniques—Accelerated Solvent Extraction (ASE) and Solid Phase Extraction (SPE)—were used to obtain metabolite-rich fractions. Metabolite profiling via UPLC-QTOF-MSE identified 29 compounds, of which 15 were confidently annotated with the support of 1D and 2D NMR data.
Multivariate analysis using OPLS-DA, based on UPLC-QTOF-MSE data, revealed a clear chemical distinction between the ASE and SPE extracts, with robust model statistics (R²Y and Q² > 0.94). Variable Importance in Projection (VIP) scores highlighted key metabolites responsible for the observed biological activity, particularly flavonoids (in SPE) and anacardic acids (AAs) (in ASE), both of which showed strong correlation with α-AMY inhibition. These compounds were further evaluated using molecular docking (CB-Dock2 and Swiss Dock platform), which predicted strong binding affinities at the enzyme’s active site (PDB ID: 2QV4), consistent with the docking profile of acarbose, the reference inhibitor. In vitro enzymatic assays confirmed the in silico results: the ASE extract exhibited greater α-AMY inhibitory activity (IC50= 40.80 ± 0.60 µg/mL) compared to the SPE extract (IC50 = 59.43 ± 2.63 µg/mL), and both were significantly more potent than acarbose (IC₅₀ = 208.4 ± 0.31 µg/mL).
The extraction method significantly influenced both the chemical composition and the biological efficacy of the extracts. The SPE extract was enriched with flavonolssuch as rutin, quercetin, and kaempferol, while the ASE extract was dominated by various anacardic acids ((17:3), (15:3), (15:2), and (15:1)-AA), all of which demonstrated high binding affinity to α-AMY.
Importantly, this study also aimed to validate and propose a structured pipeline for targeted discovery of enzyme inhibitors from natural products, integrating metabolomics, chemometric modeling, and molecular docking. Ongoing assays with commercial standards of key compounds aim to further confirm their role as bioactive markers.
In summary, the findings support S. tuberosa as a promising natural source of α-AMY inhibitors and highlight the value of integrated analytical strategies in the discovery of plant-derived agents for glycemic control in T2D.
Agradecimentos: Agradecemos à FIOCRUZ, EMBRAPA, UNILAB, UFSC, CAPES e CNPq pelo apoio essencial ao desenvolvimento deste projeto. Suas contribuições, por meio de infraestrutura, colaboração científica e financiamento, foram fundamentais para a realização das análises e avanços obtidos neste estudo.