Anti-oxidant and anti-inflammatory activities of a flavonoid-rich extract from Citrus bergamia Risso et Poiteau juice in both cell-free and in vitro models

Inflammation consists in a series of biological reactions induced by the alteration of tissue homeostasis occurring in response to biological, chemical or physical agents in the organism. Oxidative stress is defined as an imbalance between production and elimination of free radicals and reactive metabolites (oxidants). This is a natural physiological process where the presence of ROS overpowers the cellular radical scavenging ability, thus creating an imbalance in the oxidative status between the oxidants and the anti-oxidants. Inflammation and oxidative stress are closely related pathophysiological events that are strongly linked each other. One of them may appear before or after the other, but when one emerges the other one is most likely to follow, and then both of them take part in the pathogenesis of many disorders. In recent years, there has been an extraordinary increase in the number of studies on anti-oxidant properties of various phytochemicals, able to counteract reactive species (RS) overproduction. Among these, flavonoids have been extensively studied mainly for their anti-oxidant property which ameliorates many inflammatory diseases and was linked to the maintenance of good health. Citrus fruits and their juices are the main food sources of flavonoids which exert protective effects against numerous degenerative processes. In the last decade a number of studies have investigated the anti-oxidant and anti-inflammatory effects of each single Citrus flavonoid as pure compounds. However, few studies have focused on the pharmacological activity of Citrus juices and extracts and its molecular mechanisms underlying their potential beneficial effects. On these bases during my PhD, I focused on the anti-oxidant activity of a flavonoid-rich extract from Citrus bergamia juices (BJe) and its effect against inflammatory processes. First, we tested the anti-oxidant properties of BJe in cell-free experimental models by ORAC, DPPH, Folin-Ciocalteu and Reducing Power assays, proving its anti-oxidant activity. Then, we assayed its ability to prevent the cytotoxic effects induced by H2O2 or Fe2(SO4)3. Our results provided evidences that BJe reduces cell death, generation of ROS and membrane lipid peroxidation, improve mitochondrial functionality and prevents DNA-oxidative damage in A549 cells incubated with H2O2. Moreover, BJe is able to both induce catalase expression and increase its activity. Furthermore, evidences, that BJe prevents the cytotoxic effects by Fe2(SO4)3, have suggested that this extract could possess chelating properties. This hypothesis was confirmed by measuring the presence of redox-active iron in the cells pre-treated with the extract and then exposed to the metal. In the light of these observations, we wondered whether BJe may be effective against inflammatory processes. To this aim, we used THP-1 monocytes to investigate the mechanisms underlying the beneficial potential of BJe against two different models of inflammation in which the THP-1 cells were exposed to LPS or amyloid-beta1–42 (Aβ1−42). Exposure of THP-1 cells to BJe inhibited both gene expression and secretion of LPS-induced pro-inflammatory cytokines (IL-6, IL-1β, TNF-α) by a mechanism involving the inhibition of NF-ĸB activation. In addition, BJe treatment reversed the LPS enhanced acetylation of p65 in THP-1 cells. Furthermore, increasing concentrations of Sirtinol were able to suppress the inhibitory effect of BJe via p65 acetylation,underscoring that NF-ĸB-mediated inflammatory cytokine production may be directly linked to SIRT1 activity.Treatment of THP-1 cells with Aβ1−42 significantly induced the expression and secretion of IL-6 and IL-1β in THP-1 cells and increased the phosphorylation of ERK 1/2 as well as p46 and p54 members of JNK family. Moreover, Aβ1−42 raised AP-1 DNA binding activity in THP-1-treated cells. Interestingly, all these effects were reduced in the presence of BJe. In conclusion, our studies provided evidences that BJe may be used in preventing oxidative cell injury suggesting a promising role as a natural drug against inflammatory processes. In addition, the complex mixture of phytochemicals present in the whole extract acts better than the single constituent. This is because all molecules present in a phytocomplex can modulate simultaneously different targets of action in both human cells and microorganisms, leading to a pool of pharmacological effects contributing together to improve patient’s health.