Multi-targeted MS-based metabolomics fingerprinting of black and white pepper coupled with molecular networking in relation to their <i>in vitro</i> antioxidant and antidiabetic effects

Spices are considered as a valuable food material owing not only to their special aroma, but also a myriad of nutritional and health benefits. Black pepper (<i>Piper nigrum</i> L.; Piperaceae) is known as the "king of spices", being commonly used worldwide in its two forms: black and processed white pepper. The main goal of this study was to perform multi-targeted comparative metabolite profiling and fingerprinting approaches targeting primary and secondary metabolites using gas chromatography mass-spectrometry (GC-MS) post-silylation and ultra-performance liquid chromatography (UPLC-MS/MS) coupled to multivariate analyses and molecular networking. A total of 51 metabolites were annotated using GC-MS belonging to fatty acids/esters (9), alkaloids/nitrogenous (6), sugars (3), sugar alcohols (5), organic acids (15), alcohols (4), and aliphatic hydrocarbons (6) in addition to phenols (3). Fatty acids/esters were enriched in black and white pepper at <i>ca.</i> 23.4 mg g^-1. Moreover, piperine was detected at higher levels in white pepper at 5.9 mg g^-1 compared to 3.4 mg g^-1 in black pepper. A total of 71 metabolites were annotated using UPLC-MS/MS, with piperamides as the most abundant class, of which 6 are first time to be detected in <i>P. nigrum</i> fruit "types A, E and O". In addition, 7 fatty acids were recoded along 4 flavonoids exhibiting novel glycosidic linkage of kaempferol and apigenin. Furthermore, 5 hydroxycinnamic acids have been detected; some were identified for the first time from <i>P. nigrum</i> fruit. Clusters of fatty acids, flavonoids and phenylamides were detected by negative mode GNPS molecular networking, whereas clusters representing the majority of alkaloids were detected in positive mode. Assay of total phenolics and flavonoids revealed higher levels in black compared to white pepper, with values of 45.6 and 37.5 mg GAE per g for total phenolics and 9.4 & 8.5 mg RE per g for flavonoids, respectively. Assessment of antioxidant capacity using DPPH, ABTS scavenging assays, and FRAP assay revealed moderate effects at 49.79, 20.6, and 104.6 (black pepper), 29.0, 11.5, and 77.5 mg TE per g (white pepper), respectively. Moreover, black and white pepper extracts inhibited α-glucosidase enzyme with an IC₅₀ of 0.77 and 0.62 mg mL^-1, compared with acarbose.