Antioxidant, Anti-Inflammatory and Anti-Angiogenic Properties of Citrus lumia Juice

Abstract

Citrus juices are a rich source of bioactive compounds with various and well-known health benefits. The aim of this study was to investigate the polyphenols and ascorbic acid content as well as to investigate the antioxidant, anti-inflammatory and anti-angiogenic properties of the juice of an ancient Mediterranean species, Citrus lumia Risso (CLJ). The antioxidant and anti-inflammatory activities were evaluated by several in vitro cell-free and cell-based assays, whereas two different in vivo models, the chick chorioallantoic membrane (CAM) and the zebrafish embryos, were used to characterize the anti-angiogenic properties. Twenty-eight polyphenols were identified by RP-LC-DAD-ESI-MS analysis (flavonoids 68.82% and phenolic acids 31.18%) with 1-caffeoyl-5-feruloylquinic acid and kaempferol 3'-rhamnoside, which represent the most abundant compounds (25.70 and 23.12%, respectively). HPLC-DAD analysis showed a high ascorbic acid content (352 mg/kg of CLJ), which contributes with polyphenols to the marked and dose-dependent antioxidant and anti-inflammatory properties observed. CLJ showed strong and dose-dependent anti-angiogenic activity as highlighted by the inhibition of blood vessel formation on CAMs and the decrease of endogenous alkaline phosphatase on zebrafish embryos. Moreover, within the concentration range tested, no dead or malformed embryos were recorded. Certainly, further studies are needed to investigate the molecular mechanisms underlying these promising biological effects, but considering the evidence of the present study, the use of CLJ as a ready-to drink safe prevention strategy for inflammatory-based diseases correlated to angiogenesis could be justified.

Keywords: anti-angiogenic; anti-inflammatory; antioxidant; ascorbic acid; chick chorioallantoic membrane; citrus juice; polyphenols; zebrafish embryos.

FIGURE 1

Representative LC-DAD chromatogram of CLJ acquired at 330 nm (A). Peak numbers refer to compounds listed in Table 1 according to their elution order. Panel B reports the composition of CLJ in terms of polyphenols classes expressed as mean area percentage with respect to all polyphenols identified.

FIGURE 2

Antioxidant and free radical-scavenging activity of several concentrations of CLJ towards DPPH (A); TEAC (B); FRAP (C); ORAC (D); Iron-chelating activity (E) and β-carotene bleaching (F) assay. Results were expressed as mean inhibition percentage ± standard deviation of three independent experiments (n = 3).

FIGURE 3

Anti-inflammatory activity of several concentrations of CLJ evaluated by BSA denaturation (A) and anti-protease activity (B) assays. Diclofenac sodium (50 and 80 µg/ml, respectively) was used as reference compound in both assays. Results were expressed as mean inhibition percentage ± standard deviation of three independent experiments (n = 3). *p < 0.01 vs positive control (CTR+).

FIGURE 4

Evaluation of antioxidant and anti-inflammatory activity of CLJ by cell-based assays carried out on erythrocytes (A,B) and PBMC (C,D). (A) Heat-induced hemolysis, diclofenac sodium 50 µg/ml was used as positive control (CTR+); (B) Antioxidant activity against intracellular ROS, trolox 50 µg/ml was used as positive control (CTR+); (C,D) IL-6 and TNF-α release by PBMC after LPS-induced inflammation, diclofenac sodium 50 µg/ml was used as positive control (CTR+) whereas cell medium culture containing 0.1% DMSO was used as negative control (CTR-). *p < 0.001 vs CTR-; ^§ p < 0.001 vs CTR+.

FIGURE 5

Anti-angiogenic properties of several concentrations of CLJ evaluated by CAM assay (A). In (B), representative microscopic images of the CAMs after treatment with the reference compound (retinoic acid, 10 µg/ml) (A), 700–90 µg/ml CLJ samples (B-E) and negative control (deionized water) (F) are reported. Results were expressed as mean inhibition percentage ± standard deviation of three independent experiments (n = 5). *p < 0.01 vs positive control (CTR+) retinoic acid 10 µg/ml.

FIGURE 6

Anti-angiogenic properties of several concentrations of CLJ (125–500 µg/ml) evaluated by EAP activity of vascular endothelial cells released from treated zebrafish embryos (A). (B) shows a microscopic image of Zebrafish embryo (magnification 20×). Results were expressed as mean inhibition percentage ± standard deviation of three independent experiments (n = 5). *p < 0.01 vs positive control (CTR+) 2-methylestradiol 30 µg/ml.