Characterization of Health Beneficial Components in Discarded Leaves of Three Escarole (Cichorium endivia L.) Cultivar and Study of Their Antioxidant and Anti-Inflammatory Activities

Abstract

Plants of genus Cichorium (Asteraceae) can be used as vegetables with higher nutritional value and as medicinal plants. This genus has beneficial properties owing to the presence of a number of specialized metabolites such as alkaloids, sesquiterpene lactones, coumarins, unsaturated fatty acids, flavonoids, saponins, and tannins. Cichorium endivia L., known as escarole, has achieved a common food status due to its nutritionary value, bitter taste, and the presence of healthy components, and is eaten cooked or raw in salads. Presently, wastes derived from the horticultural crops supply chain are generated in very large amounts. Vegetable waste comprises the discarded leaves of food sources produced during collection, handling, transportation, and processing. The external leaves of Cichorium endivia L. are a horticultural crop that is discarded. In this work, the phytochemical profile, antioxidant, and anti-inflammatory activities of hydroalcoholic extract obtained from discarded leaves of three cultivars of escarole (C. endivia var. crispum 'Capriccio', C. endivia var. latifolium 'Performance' and 'Leonida') typical horticultural crop of the Campania region were investigated. In order to describe a metabolite profile of C. endivia cultivars, the extracts were analysed by HR/ESI/Qexactive/MS/MS and NMR. The careful analysis of the accurate masses, the ESI/MS spectra, and the ¹H NMR chemical shifts allowed for the identification of small molecules belonging to phenolic, flavonoid, sesquiterpene, amino acids, and unsaturated fatty acid classes. In addition, the antioxidant potential of the extracts was evaluated using cell-free and cell-based assays, as well as their cytotoxic and anti-inflammatory activity. All the extracts showed similar radical-scavenging ability while significant differences between the three investigated cultivars emerged in the cell-based assays. The obtained data were ascribed to the content of polyphenols and sesquiterpenes in the extracts. Accordingly, C. endivia by-products can be deemed an interesting material for healthy product formulations.

Keywords: Cichorium endivia L.; HRESIMS; NMR; anti-inflammatory; antioxidant; cultivar; green extraction; waste valorisation.

Figure 1

The effect of power ultrasound on C. endivia cultivars leaves. Comparison of chlorophyll extraction kinetics for UAE, US probe 25 kHz and 40 kHz, and for maceration.

Figure 2

¹H NMR spectrum of C. endivia cultivars from δ_H 0.7 to 8.5 ppm.

Figure 3

Representative ¹H NMR spectrum of C. endivia ‘Performance’ extract with annotation of the metabolites of the Chenomx 600 MHz custom library (CCL). Regions δ H 0.9–3.1, δ H 3.0–5.5, and δ H 5.6–8.5 were expanded.

Figure 4

LC-MS/MS-based quantitative analysis of the sesquiterpene lactones retrieved in the leaf extracts of the three cultivars of C. envidia. The sesquiterpenes were quantified as lactucin.

Figure 5

LC-MS/MS-based quantitative analysis of the kaempferol derivatives (KMHex: kaempferol-O-malonylhexoside, KHex: kaempferol-O-hexoside, KMHex2: kaempferol-O-malonylhexoside isomer, KHexU: kaempferol-O-hexoside-uronide, KdHex: kaempferol-O-dihexoside, KAHex: kaempferol-O-acetylhexoside, KU: kaempferol-O-uronide) retrieved in the leaf extracts of the three cultivars of C. envidia. The compounds were quantified as kaempferol.

Figure 6

C. endivia leaves extracts’ H₂O₂ scavenging activity. H₂O₂ concentration measured through HRP enzymatic assay after incubation with different concentrations of C. endivia extracts is shown. Positive control is represented by the enzymatic assay in the absence of natural extracts and negative control was performed with a previous incubation of the reaction mixture with catalase to obtain H₂O₂ degradation.

Figure 7

(A) Cellular antioxidant activity (CAA) of ‘Leonida’, ‘Performance’, and ‘Capriccio’ on THP-1-derived macrophage M0 cells at the concentration of 100 μg/mL. (B) Cellular Antioxidant Activity (CAA) of C. endivia cultivars ‘Leonida’ on THP-1-derived macrophage M0 cells at the concentration of 50–100 μg/mL.

Figure 8

Effect of ‘Leonida’, ‘Performance’, and ‘Capriccio’ extracts on SOD enzymatic activity in THP-1-derived macrophage M0 cells at a concentration of 100 μg/mL.

Figure 9

Western blot of ‘Leonida’, ‘Performance’, and ‘Capriccio’ tested at the concentration of 50–100 µg/mL on THP-1-derived M0 in the presence of Free Radical Initiator.

Figure 10

(A) Percentage of secretion of IL-6 from LPS-stimulated THP-1-derived macrophage M0 cells after 24 h of treatment with ‘Leonida’, ‘Performance’, and ‘Capriccio’ at the concentration of 100 μg/mL. (B) Percentage of secretion of IL-6 from LPS-stimulated THP-1-derived macrophage M0 cells after 24 h of treatment with ‘Leonida’ at the concentration of 50 and 100 μg/mL.