Influence of Maturity Stage and Sampling Time on the Metabolite Profiles of Piper sarmentosum Using Optimized NMR Parameters

Optimization is a critical step in metabolomics workflows to ensure reliable data, particularly when handling large sample numbers. In this study, optimization was carried out for Piper sarmentosum Roxb., a medicinal herb of regional importance, to establish standardized procedures for NMR-based metabolomic analysis and sample collection. Extraction conditions were evaluated using different sample masses, with 75 mg of freeze-dried material providing the best signal intensity, particularly in the aromatic region. Several NOESY-based NMR parameter sets were then compared, and the settings adapted from Halabalaki et al. (2014) produced the highest spectral quality and were adopted for further analyses. Following optimization, the influence of leaf maturity and harvesting time on metabolite composition was assessed using 1D NOESY NMR spectroscopy. Partial least square discriminant analysis (PLS-DA) revealed clear metabolic distinctions between young and mature leaves, while samples collected in the morning and afternoon showed no notable differences, indicating that sampling time had minimal impact on metabolite composition. The optimized protocols established in this work minimize technical variability, enhance spectral reproducibility, and improve metabolite detectability. These outcomes provide a robust platform for large-scale metabolomics investigations of P. sarmentosum and other medicinal plants.