Inflammation and Obesity: The Pharmacological Role of Flavonoids in the Zebrafish Model.

A Mediterranean-style diet is highly encouraged thanks to its healthy food pattern, which includes valuable nutraceuticals such as polyphenols. Among these, flavonoids are associated with relevant biological properties through which they prevent or fight the onset of several human pathologies. Globally, the enhanced incidence of overweight and obese people has caused a dramatic increase in comorbidities, raising the need to provide better therapies. Therefore, the development of sophisticated animal models of metabolic dysregulation has allowed for a deepening of knowledge on this subject. Recent advances in using zebrafish (Danio rerio) as model for metabolic disease have yielded fundamental insights into the potential anti-obesity effects of flavonoids. Chronic low-grade inflammation and immune system activation seem to characterize the pathogenesis of obesity; thus, their reduction might improve the lipid profile of obese patients or prevent the development of associated metabolic illnesses. In this review, we highlight the beneficial role of flavonoids on obesity and related diseases linked to their anti-inflammatory properties. In light of the summarized studies, we suggest that anti-inflammatory therapies could have a relevant place in the prevention and treatment of obesity and metabolic disorders.

Oleacein Attenuates Lipopolysaccharide-Induced Inflammation in THP-1-Derived Macrophages by the Inhibition of TLR4/MyD88/NF-κB Pathway.

It is known that plant phenolic compounds exert anti-inflammatory activity through both anti-oxidant effects and modulation of pivotal pro-inflammatory factors. Recently, Olea europaea has been studied as a natural source of bioactive molecules; however, few studies have focused on the biological effect of oleacein (OLC), the most abundant secoiridoid. Therefore, the aim of this study was to investigate the potential anti-oxidant activity of OLC, as well as to study its anti-inflammatory effect in lipopolysaccharide (LPS)-stimulated THP-1-derived macrophages. LPS brought a dramatic increase of both release and gene expression of pro-inflammatory cytokines (IL-6, IL-1ß and TNF-α), as well as a decrease of anti-inflammatory ones (IL-10), the effects of which are reverted by OLC. Moreover, it reduced the levels of COX-2, NO and PGE2 elicited by LPS exposure in THP-1 macrophages. Interestingly, OLC modulated inflammatory signaling pathways through the inhibition of CD14/TLR4/CD14/MyD88 axis and the activation of NF-κB. Finally, OLC showed relevant anti-oxidant capability, assessed by abiotic assays, and reduced the intracellular amount of ROS generated by LPS exposure in THP-1 macrophages. Overall, these results suggest that the anti-oxidant activity and anti-inflammatory effect of OLC may cooperate in its protective effect against inflammatory stressors, thus being a possible alternative pharmacological strategy aimed at reducing the inflammatory process.

The Second Life of Citrus Fruit Waste: A Valuable Source of Bioactive Compounds.

Citrus fruits (CF) are among the most widely cultivated fruit crops throughout the world and their production is constantly increasing along with consumers' demand. Therefore, huge amounts of waste are annually generated through CF processing, causing high costs for their disposal, as well as environmental and human health damage, if inappropriately performed. According to the most recent indications of an economic, environmental and pharmaceutical nature, CF processing residues must be transformed from a waste to be disposed to a valuable resource to be reused. Based on a circular economy model, CF residues (i.e., seeds, exhausted peel, pressed pulp, secondary juice and leaves) have increasingly been re-evaluated to also obtain, but not limited to, valuable compounds to be employed in the food, packaging, cosmetic and pharmaceutical industries. However, the use of CF by-products is still limited because of their underestimated nutritional and economic value, hence more awareness and knowledge are needed to overcome traditional approaches for their disposal. This review summarizes recent evidence on the pharmacological potential of CF waste to support the switch towards a more environmentally sustainable society.

Moringin from Moringa Oleifera Seeds Inhibits Growth, Arrests Cell-Cycle, and Induces Apoptosis of SH-SY5Y Human Neuroblastoma Cells through the Modulation of NF-κB and Apoptotic Related Factors.

In the last decades, glucosinolates (GLs), precursors of isothiocyanates (ITCs), have been studied mostly for their chemopreventive and chemotherapeutic properties. The aim of our research was to study the antiproliferative effect of 4-(α-L-rhamnopyranosyloxy) benzyl glucosinolate (glucomoringin; GMG) bioactivated by myrosinase enzyme to form the corresponding isothiocyanate 4-(α-L-rhamnopyranosyloxy) benzyl C (moringin) in SH-SY5Y human neuroblastoma cells. We found that moringin significantly reduced SH-SY5Y cell growth in a time and concentration-dependent (p < 0.05, 0.01, and 0.001 vs. ctrl, after treatment with 16.4 µM moringin for 24, 48, and 72 h, respectively) manner through a mechanism involving the activation of apoptotic machinery. In addition, it altered the normal progression of cells through the cell cycle, increasing the cell population in both G2 and S phases, as well as decreasing that in the G1 phase. Studying the drug mechanism of action, we found that moringin was able to increase the expression of p53, p21, and Bax at both the protein and transcriptional level. Moreover, exposure of SH-SY5Y cells to moringin significantly increased the gene expression of both caspase 3 and 9 and enhanced their cleavage, thereby initiating an intrinsic apoptotic cascade. Finally, moringin inhibited nuclear translocation of NF-κB. Our study demonstrates the ability of moringin to reduce the growth of SH-SY5Y cells and reveals its mechanism of action, suggesting its promising role as an anticancer drug.

Citrus Flavonoids and Autoimmune Diseases: A Systematic Review of Clinical Studies.

BACKGROUND: Autoimmune diseases are chronic disorders in which the immune system does not recognize and attacks one self's healthy components. In this context, although natural remedies might represent a promising therapeutic strategy, evidence regarding Citrus flavonoids is still controversial. OBJECTIVE: To summarize and critically discuss the clinical evidence on the effects of Citrus flavonoids on managing autoimmune diseases. METHOD: A systematic review of articles has been carried out independently by two authors using MEDLINE, Scopus and ISI Web of Science databases. Search terms comprised keywords related to Citrus flavonoids and autoimmune diseases. The last search was performed on the 16th of March, 2021. No language restrictions were applied. Systematic review and study selection were conducted in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement. Before starting the review, the authors defined the types of articles to be included. Three reviewers independently carried out the extraction of papers. RESULTS: Ten clinical studies fulfilled the eligibility criteria and were included in the final review. CONCLUSION: The studies discussed in this review are heterogeneous. Indeed, some studies suggest using Citrus flavonoids in the frame of autoimmune disorders, whereas others discourage it. Hence, this systematic review highlights the need for further large-scale clinical studies to define the exact role of Citrus flavonoids in managing autoimmune diseases (PROSPERO number CRD42021234903).

The Anticancer Effect of a Flavonoid-Rich Extract of Bergamot Juice in THP-1 Cells Engages the SIRT2/AKT/p53 Pathway.

Novel targets are constantly sought to fight hematologic malignancies. In this regard, high levels of SIRT2 expression are associated with unfavorable prognosis of acute myeloid leukemia. The interest in the plant kingdom has allowed the identification of ever-new anti-leukemic agents. Citrus × bergamia (bergamot) was proved to possess anticancer properties, yet no evidence is available regarding leukemia. For the first time, we studied the potential anti-leukemic effect of a flavonoid-rich extract of bergamot juice (BJe) in THP-1 cells, investigating the underlying mechanisms. Our findings showed that BJe reduced THP-1 cell proliferation, without affecting that of primary PBMCs, blocking the cell cycle in S phase and inducing apoptosis. Triggering of both extrinsic and intrinsic apoptotic pathways was witnessed by cleavage of caspase-8 and -9, which in turn activated caspase-3 and PARP. Interestingly, the increased p53 acetylation in THP-1 cells underlies SIRT2 inhibition by BJe, that was proved also in the isolated enzyme. Moreover, BJe hampered SIRT2 also by lowering its gene expression. Finally, BJe reduced AKT phosphorylation, which we hypothesized being the joining link between SIRT2 and p53, that play a pivotal role in BJe-induced cell cycle arrest and apoptosis in THP-1 cells. Our results suggest BJe as a potential anti-leukemic agent, via targeting of the SIRT2/AKT/p53 pathway.

The SIRT2 Pathway Is Involved in the Antiproliferative Effect of Flavanones in Human Leukemia Monocytic THP-1 Cells.

Acute myeloid leukemia (AML) represents the most alarming hematological disease for adults. Several genetic modifications are known to be pivotal in AML; however, SIRT2 over-expression has attracted the scientific community's attention as an unfavorable prognostic marker. The plant kingdom is a treasure trove of bioactive principles, with flavonoids standing out among the others. On this line, the aim of this study was to investigate the anti-leukemic properties of the main flavanones of Citrus spp., exploring the potential implication of SIRT2. Naringenin (NAR), hesperetin (HSP), naringin (NRG), and neohesperidin (NHP) inhibited SIRT2 activity in the isolated recombinant enzyme, and more, the combination between NAR and HSP. In monocytic leukemic THP-1 cells, only NAR and HSP induced antiproliferative effects, altering the cell cycle. These effects may be ascribed to SIRT2 inhibition since these flavonoids reduced its gene expression and hampered the deacetylation of p53, known sirtuin substrate, and contextually modulated the expression of the downstream cell cycle regulators p21 and cyclin E1. Additionally, these two flavanones proved to interact with the SIRT2 inhibitory site, as shown by docking simulations. Our results suggest that both NAR and HSP may act as anti-leukemic agents, alone and in combination, via targeting the SIRT2/p53/p21/cyclin E1 pathway, thus encouraging deeper investigations.

A Flavonoid-Rich Extract of Mandarin Juice Counteracts 6-OHDA-Induced Oxidative Stress in SH-SY5Y Cells and Modulates Parkinson-Related Genes.

Parkinson's disease (PD) is a degenerative disorder of the nervous system due to unceasing impairment of dopaminergic neurons situated in the substantia nigra. At present, anti-PD drugs acting on dopamine receptors are mainly symptomatic and have only very limited neuroprotective effects, whereas drugs slowing down neurodegeneration of dopaminergic neurons and deterioration of clinical symptoms are not yet available. Given that, the development of more valuable pharmacological strategies is highly demanded. Comprehensive research on innovative neuroprotective drugs has proven that anti-inflammatory and antioxidant molecules from food sources may prevent and/or counteract neurodegenerative diseases, such as PD. The present study was aimed at the evaluation the protective effect of mandarin juice extract (MJe) against 6-hydroxydopamine (6-OHDA)-induced SH-SY5Y human neuroblastoma cell death. Treatment of differentiated SH-SY5Y cells with 6-OHDA brought cell death, and specifically, apoptosis, which was significantly inhibited by the preincubation with MJe through caspase 3 blockage and the modulation of p53, Bax, and Bcl-2 genes. In addition, it showed antioxidant properties in abiotic models as well as in vitro, where it reduced both reactive oxygen and nitrogen species induced by 6-OHDA, along with restored mitochondrial membrane potential, and prevented the oxidative DNA damage evoked by 6-OHDA. Furthermore, MJe restored the impaired balance of SNCA, LRRK2, PINK1, parkin, and DJ-1 gene levels, PD-related factors, caused by 6-OHDA oxidative stress. Overall, these results indicate that MJe exerts neuroprotective effects against 6-OHDA-induced cell death in SH-SY5Y cells by mechanisms involving both the specific interaction with intracellular pathways and its antioxidant capability. Our study suggests a novel possible strategy to prevent and/or ameliorate neurodegenerative diseases, such as PD.

Anti-inflammatory effect of a flavonoid-rich extract of orange juice in adult zebrafish subjected to Vibrio anguillarum-induced enteritis.

Inflammation-related pathologies remain a serious health problem with high costs for the community. Citrus flavonoids are known to possess important pharmacological properties, including anti-inflammatory activity. In this study we evaluated the effects of a flavonoid-rich extract of orange juice (OJe) in an experimental model of enteritis induced by Vibrio anguillarum in adult zebrafish (Danio rerio). Administration of V. anguillarum through live feed (Artemia nauplii) for three consecutive days caused evident signs of enteritis in zebrafish. Three days of treatment with OJe before the pathogenic insult resulted in a remarkable reduction of tissue inflammatory events as well as a molecular down-regulation of the inflammatory genes such as IL-1ß, IL-6 and TNFα. Our data suggest that OJe counteracts the inflammation of zebrafish intestinal mucosa, indicating that the pool of flavonoids present in orange juice could be useful for the prevention of enteritis.

Bergamottin and 5-Geranyloxy-7-methoxycoumarin Cooperate in the Cytotoxic Effect of Citrus bergamia (Bergamot) Essential Oil in Human Neuroblastoma SH-SY5Y Cell Line.

The plant kingdom has always been a treasure trove for valuable bioactive compounds, and Citrus fruits stand out among the others. Bergamottin (BRG) and 5-geranyloxy-7-methoxycoumarin (5-G-7-MOC) are two coumarins found in different Citrus species with well-acknowledged pharmacological properties. Previously, they have been claimed to be relevant in the anti-proliferative effects exerted by bergamot essential oil (BEO) in the SH-SY5Y human neuroblastoma cells. This study was designed to verify this assumption and to assess the mechanisms underlying the anti-proliferative effect of both compounds. Our results demonstrate that BRG and 5-G-7-MOC are able to reduce the proliferation of SH-SY5Y cells, inducing apoptosis and increasing cell population in sub-G0/G1 phase. Moreover, we demonstrated the pro-oxidant activity of the two coumarins that increased reactive oxygen species and impaired mitochondrial membrane potential. From a molecular point of view, BRG and 5-G-7-MOC were able to modulate apoptosis related factors at both protein and gene levels. Lastly, we evaluated the synergistic effect of their combination, finding that the highest synergy was observed at a concentration ratio similar to that occurring in the BEO, supporting our initial hypothesis. Taken together, our results deepen the knowledge regarding the effect of BRG and 5-G-7-MOC in SH-SY5Y cells, emphasizing the relevance of their cooperation in achieving this effect.

Mechanisms Underlying the Anti-Inflammatory Activity of Bergamot Essential Oil and Its Antinociceptive Effects.

Renewed interest in natural products as potential source of drugs led us to investigate on both the anti-inflammatory and anti-nociceptive activity of Citrus bergamia Risso et Poiteau (bergamot) essential oil (BEO). Carrageenan-induced paw edema in rats was used as an experimental model of inflammation. Because of the toxicity of furocoumarins, we performed our study by using the BEO fraction deprived of these compounds (BEO-FF). Treatment with BEO-FF led to a significant inhibition of paw edema induced by a sub-plantar injection of carrageenan. Moreover, histological examination of BEO-FF-treated rat paw biopsies showed a reduction of pathological changes typical of edema. Pre-treatment with BEO-FF significantly reduced interleukin (IL)-1ß, IL-6, and tumor necrosis factor (TNF)-α levels in the paw homogenates, as well as nitrite/nitrate and prostaglandin E2 (PGE2) content in exudates. In addition, BEO-FF possesses antioxidant properties, as determined by cell-free assays. Furthermore, results of the writhing test showed that BEO-FF elicited a pronounced analgesic response, as demonstrated by a significant inhibition of constrictions in mice receiving acetic acid, with respect to control animals, whereas the results of the hot plate test suggested that the supra-spinal analgesia participates in the anti-nociceptive effect of BEO-FF. Our study indicates that BEO-FF exerts anti-inflammatory and anti-nociceptive effects, and suggests its potential role as an anti-edemigen and analgesic drug.

Citrus fruits and their flavonoids in inflammatory bowel disease: an overview.

Inflammatory bowel disease (IBD), with its major manifestations being Crohn's disease and ulcerative colitis, belongs to the gastrointestinal inflammatory disorders, whose main therapeutic approach is represented by synthetic anti-inflammatory drugs. However, they are often accompanied by many side effects that shifted the interest of the scientific community towards natural products. In this context, several studies asserted the anti-IBD effects of Citrus fruits and their flavonoids, thus the aim of the present review is to provide robust evidence favouring their role in the prevention and treatment of IBD. Key mechanisms relate to their anti-inflammatory and antioxidant properties, as well as their ability to modulate gut microbiota. All the findings collected in this review, lay the foundations for further studies in human with the aim of evaluating the concrete applicability as a novel preventive and therapeutic approach of Citrus fruits and their flavonoids.[Figure: see text].