The structural characterization of a new flavonoid from bergamot fruit (Citrus bergamia Risso) carrying the 3-hydroxy-3-methyl glutaryl (HMG) ester moiety has been accomplished, and its antioxidant ability was tested from a chemical point of view. The peculiarity of the new molecule, named peripolin, relies on the presence of the HMG chemical group linked to the sugar portion of neoeriocitrin; the structure was elucidated using both high-resolution mass spectrometry and nuclear magnetic resonance experiments performed on the purified molecule extracted from the fruit. The antioxidant ability of the new molecule was tested by classical chemical approaches, such as DPPH, ABTS and FRAP assays, and from a theoretical point of view. ¹H and ¹³C NMR experiments and HR-ESI-MS/MS experiments show unequivocally that the HMG moiety is linked to the primary position of the glucose unit of neohesperidose, while the chemical tests and the computational results show that peripolin possesses strong antioxidant behavior, similar to that of neoeriocitrin and remarkably higher respect to the other flavonoids present in the fruit. Furthermore, the quantitative assays carried out by UPLC-MS/MS showed that its amount in the fruit is similar to that of the other main flavonoids. Furthermore, molecular dynamics simulations allowed us to investigate the possible conformations adopted by the antioxidants in the presence of water molecules. In particular, the switch of open-closed conformations of HMG-containing species was evidenced. As far as the reaction with DPPH, the calculation of ΔG_rea supported the experimental outcomes regarding the peripolin and neoeriocitrin activity. In conclusion, bergamot fruit, already known for its potential to lower the level of blood cholesterol, has been proven to contain molecules such as neoeriocitrin and the newly characterized peripolin, which could have important in-vivo antioxidant characteristics.

With the aim of characterising the hypo‐lipidemic function of the Brumex™ ingredient obtained from the whole fruit of Citrus bergamia, a combined pre‐clinical and clinical study was conducted. In the HepG2 experimental model, we first demonstrated that Brumex™ does not trigger any significant alteration in cell viability over the tested concentration range of 1–2000 μg/mL (4 and 24 h). By stimulating the phosphorylation of AMP‐activated protein kinase (AMPK) at threonine 172, Brumex™ significantly reduces both cholesterol and triglyceride (TG) intracellular content of HepG2 cells and impairs the expression levels of lipid synthesis‐related genes (namely, SREBF1c, SREBF2, ACACA, SCD1, HMGCR and FASN). In vitro data have been validated in a dedicated double‐blind, placebo‐controlled, randomised clinical trial performed in 50 healthy moderately hyper‐cholesterolemic subjects, undergoing supplementation with either Brumex™ (400 mg) or placebo for 12 weeks. Clinical and blood laboratory data were evaluated at the baseline and at the end of the trial. Brumex™ positively impacted on both plasma lipid pattern and liver enzymes compared with the placebo, mainly in terms of significant reduction of total cholesterol (TC), TG, low‐density lipoprotein‐cholesterol (LDL‐C), non‐high‐density lipoprotein‐cholesterol (non‐HDL‐C), apolipoprotein B100 (ApoB), fasting plasma glucose (FPG), glutamic‐oxaloacetic transaminase (GOT), glutamate pyruvate transaminase (GPT) and gamma‐glutamyl‐transferase (gGT).

Pomegranate is an important source of bioactive molecules with proven beneficial effects on human health. The aim of this study was to investigate the potential anti-inflammatory effect of a pomegranate extract (PE), obtained from the whole fruit and previously characterized by Reversed Phase-Ultra High-Pressure Liquid Chromatography-High Resolution Mass Spectrometry (RP-UHPLC–HRMS), on HepG2 human hepatocellular carcinoma cells challenged with the lipopolysaccharide (LPS). In LPS-treated cells (1 µg/ml, 24h), the PE treatment (administered at the non-cytotoxic dose of 1 µg/ml, 24h) induced a significant reduction of three key pro-inflammatory cytokines, i.e. interleukin-8 (IL-8), interleukin-1 beta (IL-1β) and tumor necrosis factor-alpha (TNF-α), at both gene expression (as assayed by real-time PCR) and secretion levels (by Enzyme-linked Immunosorbent Assay, ELISA). Although further in vivo studies are needed to prove its efficacy, this preliminary in vitro study suggests that the PE might be useful for ameliorating liver inflammation. Graphical Abstract

Pomegranate is recognized for its health benefits, primarily due to its polyphenols and metabolites, such as urolithins (Uro-A), produced via colonic fermentation of ellagic acid (EA). These compounds make pomegranate a functional food with the potential to modulate chronic disease risk factors and enhance gut health by modulating microbiota. The aims of this study were (1) to evaluate the effect of in vitro digestion and fermentation mimicking human digestive processes on the total phenolic content (TPC) and the antioxidant capacity of a standardized pomegranate extract (PE), (2) to assess the effect of the digested PE on the functionality of gut microbiota isolated from healthy and diseased subject fecal materials via short-chain fatty acid (SCFA) determination, and (3) to measure Uro-A production using UHPLC Q-Orbitrap HRMS. The in vitro digestion and fermentation processes resulted in a significant increase in the TPC, while the antioxidant capacity was considerably reduced. Following the in vitro digestion process, the TPC increased from 232 ± 16 to 1656 ± 34 g GAE/g of PE. Moreover, the TPC in the fermented samples was calculated as 6139 ± 458 g GAE/g for the microbiota of healthy adults and 8375 ± 1388 g GAE/g for the microbiota of healthy children, compared to 1657 ± 34 g GAE/g for the non-fermented samples. The PE exerted a modulatory effect on gut microbiota functionality, as reflected by an increasing concentration of SCFAs, especially lactic acid. Overall, these data suggest that pomegranate might contribute to gut health and could be a candidate for further studies in view of its possible use as a prebiotic ingredient. Further research, including clinical studies, is needed to confirm these findings and explore the potential application of pomegranate extract as a functional ingredient in nutraceuticals and functional foods aimed at improving gut health.

Fatigue, characterized by periods of exhaustion that interfere with normal activities and negatively affect quality of life, can be classified as prolonged fatigue (or short-term fatigue, SF) or chronic fatigue. Vitamin deficiency and chronic inflammation seem to be possible causes of fatigue. Due to the unavailability of effective remedies that attenuate fatigue and provide consumer satisfaction, the aim of this investigation is to assess whether a commercial food supplement consisting of pomegranate extract with known anti-inflammatory activity, B vitamins, and vitamin C can help fight SF. To this end, the chemical composition of whole fruit pomegranate dry extract was determined through a RP-UHPLC–HRMS method, which indicated the presence of ellagitannins, gallotannins, and organic and phenolic acids. In a survey of consumers of food supplements aimed at reducing fatigue, 78 subjects (21 men and 57 women) were recruited for one month to evaluate the efficacy and tolerability of this food supplement through validated questionnaires for assessing fatigue level and quality of life. The consumers reported a significant improvement in their conditions without adverse effects over this time period. Although further studies are needed, these preliminary data suggest the capacity of a combination of pomegranate extract and hydrosoluble vitamins to alleviate SF.

Prolonged fatigue is associated with non-pathological causes and lacks an established therapeutic approach. The current study is aimed at assessing the efficacy of a new food supplement (Improve™) based on a chemically characterized pomegranate extract and hydro-soluble vitamins (B complex and C). UHPLC-HRMS analysis of pomegranate extract showed the presence of 59 compounds, with gallotannins and ellagitannins being the most abundant phytochemicals. For the clinical study, 58 subjects were randomized into two groups, 1 and 2 (n = 29, each), which received either the food supplement or placebo. The effects of the food supplement against fatigue were assessed via validated questionnaires, recorded at time intervals t0 (at baseline), t1 (after 28 days), t2 (56 days), and t3 (after follow-up) in combination with the analysis of biochemical markers at t0 and t2. Fatigue severity scale (FSS) questionnaire scores were significantly decreased at the t2 and t3 time intervals in subjects treated with the food supplements, while the effect of the food supplement on a 12-Item Short Form Survey (SF-12) was not considerable. Moreover, the food supplement did not significantly affect biochemical parameters associated with fatigue and stress conditions. This study shows that the food supplement tested reduces prolonged fatigue following two months of supplementation in healthy subjects with mild prolonged fatigue.