Distinct metabolic and phytochemical signatures differentiate bergamot leaf extract from pulp by-product and support matrix-specific valorization

Abstract: Bergamot (Citrus bergamia) is cultivated predominantly in Calabria, Southern Italy, which accounts for more than 90% of global production. Its industrial value remains centered on essential oil extraction, while leaf- and pulp-derived fractions are comparatively underutilized despite their chemically diverse composition [1-3]. This study investigated whether bergamot leaf extract (BLE) and bergamot pulp by-product (BPBP) are merely quantitatively different materials or chemically distinct matrices requiring different valorization strategies. An integrated analytical approach combined untargeted proton nuclear magnetic resonance (¹H NMR) metabolomics (Varian/Agilent DD2, 499.72 MHz), reversed-phase high-performance liquid chromatography with diode-array detection (RP-HPLC-DAD) for targeted phenolic quantification, high-performance liquid chromatography (HPLC) for biogenic amine profiling, spectrophotometric determination of total phenolics, flavonoids, carotenoids, and in vitro antioxidant capacity, and ultraviolet-visible (UV-Vis) and Fourier-transform near-infrared (FT-NIR) spectroscopic fingerprinting. NMR profiling identified 89 metabolites and descriptively revealed contrasting metabolic organization. BPBP contained markedly higher concentrations of carbon-associated metabolites, including citrate (65.30 vs. 0.66 mg/g DW in BLE) and myo-inositol (26.42 vs. 1.64 mg/g DW), while fructose (27.03 mg/g DW), glucose (24.06 mg/g DW), and glucose-6-phosphate (21.23 mg/g DW) were detected in BPBP but not in BLE. Conversely, BLE showed greater representation of phenolic-related and other specialized metabolites. Replicated quantitative analyses were compared using two-tailed Welch’s t-tests. BLE contained 6.8-fold more total phenolics (p = 0.0018), 9.2-fold more flavonoids (p = 0.0019), and 7.3-fold more carotenoids (p < 0.001). DPPH, ABTS, and FRAP values were 6.7-, 7.7-, and 7.5-fold higher, respectively, in BLE. RP-HPLC confirmed higher chlorogenic acid, trans-ferulic acid, and rutin concentrations in BLE by 1.9-, 2.7-, and 10.1-fold, respectively (all p < 0.001). Total biogenic amines were 3.2-fold higher in BLE (p < 0.001), with eight of ten quantified amines significantly higher. These convergent findings indicate that the differentiation extends beyond phenolic abundance to carbon- and nitrogen-associated metabolic domains. The primary-metabolite-rich BPBP profile aligns with carbon allocation toward carbohydrate accumulation and organic acid metabolism characteristic of fruit sink tissues [4,5], whereas the specialized-metabolite- and amine-rich BLE profile aligns with broader defense, redox, and nitrogen-metabolic functions of leaves [6-8]. Distinct processing histories may also contribute to the final compositions. UV-Vis and NIR further translated these compositional differences into distinct whole-matrix optical fingerprints. Compositionally, BPBP supports evaluation for food formulation, fermentation, and biorefinery applications, whereas BLE supports investigation as a source of phenolic-rich ingredients; its higher biogenic amine content also warrants matrix-specific processing and safety assessment. Therefore, BLE and BPBP should not be regarded as richer and poorer versions of the same material, but as chemically differentiated resources supporting distinct valorization pathways.

Acknowledgments: FAPESP (2021/13050-2, 2022/01925-7, and 2023/03886-1), CNPq (306640/2023-6), and CAPES, Brazil.