Synergistic Combination of Citrus Flavanones as Strong Antioxidant and COX-Inhibitor Agent.

Recently, we demonstrated that a Citrus flavanone mix (FM) shows antioxidant and anti-inflammatory activity, even after gastro-duodenal digestion (DFM). The aim of this study was to investigate the possible involvement of the cyclooxygenases (COXs) in the anti-inflammatory activity previously detected, using a human COX inhibitor screening assay, molecular modeling studies, and PGE2 release by Caco-2 cells stimulated with IL-1ß and arachidonic acid. Furthermore, the ability to counteract pro-oxidative processes induced by IL-1ß was evaluated by measuring four oxidative stress markers, namely, carbonylated proteins, thiobarbituric acid-reactive substances, reactive oxygen species, and reduced glutathione/oxidized glutathione ratio in Caco-2 cells. All flavonoids showed a strong inhibitory activity on COXs, confirmed by molecular modeling studies, with DFM, which showed the best and most synergistic activity on COX-2 (82.45% vs. 87.93% of nimesulide). These results were also corroborated by the cell-based assays. Indeed, DFM proves to be the most powerful anti-inflammatory and antioxidant agent reducing, synergistically and in a statistically significant manner (p < 0.05), PGE2 release than the oxidative stress markers, also with respect to the nimesulide and trolox used as reference compounds. This leads to the hypothesis that FM could be an excellent antioxidant and COX inhibitor candidate to counteract intestinal inflammation.

Hesperidin Functions as an Ergogenic Aid by Increasing Endothelial Function and Decreasing Exercise-Induced Oxidative Stress and Inflammation, Thereby Contributing to Improved Exercise Performance.

The regulation of blood flow to peripheral muscles is crucial for proper skeletal muscle functioning and exercise performance. During exercise, increased mitochondrial oxidative phosphorylation leads to increased electron leakage and consequently induces an increase in ROS formation, contributing to DNA, lipid, and protein damage. Moreover, exercise may increase blood- and intramuscular inflammatory factors leading to a deterioration in endurance performance. The aim of this review is to investigate the potential mechanisms through which the polyphenol hesperidin could lead to enhanced exercise performance, namely improved endothelial function, reduced exercise-induced oxidative stress, and inflammation. We selected in vivo RCTs, animal studies, and in vitro studies in which hesperidin, its aglycone form hesperetin, hesperetin-metabolites, or orange juice are supplemented at any dosage and where the parameters related to endothelial function, oxidative stress, and/or inflammation have been measured. The results collected in this review show that hesperidin improves endothelial function (via increased NO availability), inhibits ROS production, decreases production and plasma levels of pro-inflammatory markers, and improves anaerobic exercise outcomes (e.g., power, speed, energy). For elite and recreational athletes, hesperidin could be used as an ergogenic aid to enhance muscle recovery between training sessions, optimize oxygen and nutrient supplies to the muscles, and improve anaerobic performance.

Hesperidin and Naringin Improve Broiler Meat Fatty Acid Profile and Modulate the Expression of Genes Involved in Fatty Acid ß-oxidation and Antioxidant Defense in a Dose Dependent Manner.

The beneficial properties of the flavanones hesperidin and naringin as feed additives in poultry have lately been under investigation. In broilers, both flavanones have been shown to exhibit antioxidant properties while their individual effects on fatty acid (FA) composition and the underlying molecular mechanisms of their activity have not been explored. Here, we studied their effects on broiler meats' FA profiles and on the expression of genes related to lipid metabolism, antioxidant defense and anti-inflammatory function. The experimental design comprised six treatment groups of broilers, each supplemented from day 11 until slaughter at 42 days with hesperidin, naringin or vitamin E, as follows: the E1 group received 0.75 g of hesperidin per kg of feed, E2 received 1.5 g hesperidin/kg feed, N1 received 0.75 g naringin/kg feed, N2 received 1.5 g naringin/kg feed, vitamin E (VE) received 0.2 g a-tocopheryl acetate/kg feed, and the control group was not provided with a supplemented feed. The VE treatment group served as a positive control for antioxidant activity. An analysis of the FA profiles of the abdominal adipose tissue (fat pad), major pectoralis (breast) and biceps femoris (thigh) muscles showed that both hesperidin and naringin had significant effects on saturated FA (SFA), polyunsaturated FA (PUFA) and omega n-6 content. Both compounds reduced SFA and increased PUFA and n-6 content, as well as reducing the atherogenicity and thrombogenicity indices in the breast muscle and fat pad. The effects on the thigh muscle were limited. An analysis of gene expression in the liver revealed that naringin significantly increased peroxisome proliferator-activated receptor alpha (PPARα), Acyl-CoA oxidase 1 (ACOX1) and glutathione disulfide reductase (GSR) expression. In the breast muscle, both hesperidin and naringin increased fatty acid synthase (FASN) expression and hesperidin increased the expression of adiponectin. In brief, both hesperidin and naringin supplementation beneficially affected FA profiles in the breast meat and fat pad of broiler chicken. These effects could be attributed to an increase in FA ß-oxidation since the increased expression of related genes (PPARα and ACOX1) was observed in the liver. Furthermore, the antioxidant activity of hesperidin and naringin previously observed in the meat of broilers could be attributed, at least partly, to the regulation of antioxidant defense genes, as evidenced by the increased GSR expression in response to naringin supplementation.

Four Citrus Flavanones Exert Atherosclerosis Alleviation Effects in ApoE-/- Mice via Different Metabolic and Signaling Pathways.

Citrus flavanones have the potential to alleviate atherosclerosis. The metabolism and anti-atherosclerosis signaling pathways of four citrus flavanones (naringin, naringenin, hesperidin, and hesperetin) were compared in ApoE-/- mice. Naringin had the most potent anti-atherogenic effect, followed by hesperidin, naringenin, and hesperetin with reductions of 55.92, 34.98, 42.87, and 24.70% in the atherosclerotic plaque rate compared with the control, respectively. Oral naringin mainly existed in the intestine due to the high water solubility of 7-O-nohesperidoside and alleviated atherosclerosis mainly by enhancing bile acid synthesis in the gut microbiota-FXR/FGF15-CYP7A1 pathway. The other three flavanones mainly alleviated atherosclerosis in the liver after absorption from the intestine. Hesperidin upregulates ABCA1 by 1.8-fold to enhance cholesterol reverse transport, while the aglycones naringenin and hesperetin inhibited cholesterol synthesis via downregulating HMGCR by 2.4- and 2.3-fold, respectively. Hesperetin was more resistant to absorption than naringenin due to the existence of a 4'-methoxyl group and had relatively weak effects on atherosclerosis. The alleviation of atherosclerosis by the four citrus flavanones was tightly related to differences in their in vivo metabolism and signaling pathways. This provides new insights into the anti-atherosclerotic mechanisms of food functional flavanones and guidance for the design of novel, efficient strategies for preventing atherosclerosis based on citrus flavanones.

Stratification of Volunteers According to Flavanone Metabolite Excretion and Phase II Metabolism Profile after Single Doses of 'Pera' Orange and 'Moro' Blood Orange Juices.

Large interindividual variations in the biological response to citrus flavanones have been observed, and this could be associated with high variations in their bioavailability. The aim of this study was to identify the main determinants underlying interindividual differences in citrus flavanone metabolism and excretion. In a randomized cross-over study, non-obese and obese volunteers, aged 19-40 years, ingested single doses of Pera and Moro orange juices, and urine was collected for 24 h. A large difference in the recovery of the urinary flavanone phase II metabolites was observed, with hesperetin-sulfate and hesperetin-sulfo-O-glucuronide being the major metabolites. Subjects were stratified according to their total excretion of flavanone metabolites as high, medium, and low excretors, but the expected correlation with the microbiome was not observed at the genus level. A second stratification was proposed according to phase II flavanone metabolism, whereby participants were divided into two excretion groups: Profiles A and B. Profile B individuals showed greater biotransformation of hesperetin-sulfate to hesperetin-sulfo-O-glucuronide, as well as transformation of flavanone-monoglucuronide to the respective diglucuronides, suggestive of an influence of polymorphisms on UDP-glucuronosyltransferase. In conclusion, this study proposes a new stratification of volunteers based on their metabolic profiles. Gut microbiota composition and polymorphisms of phase II enzymes may be related to the interindividual variability of metabolism.

Antioxidant and Anti-Inflammatory Activity of Citrus Flavanones Mix and Its Stability after In Vitro Simulated Digestion.

Recently, several studies have highlighted the role of Citrus flavanones in counteracting oxidative stress and inflammatory response in bowel diseases. The aim of study was to identify the most promising Citrus flavanones by a preliminary antioxidant and anti-inflammatory screening by in vitro cell-free assays, and then to mix the most powerful ones in equimolar ratio in order to investigate a potential synergistic activity. The obtained flavanones mix (FM) was then subjected to in vitro simulated digestion to evaluate the availability of the parent compounds at the intestinal level. Finally, the anti-inflammatory activity was investigated on a Caco-2 cell-based model stimulated with interleukin (IL)-1ß. FM showed stronger antioxidant and anti-inflammatory activity with respect to the single flavanones, demonstrating the occurrence of synergistic activity. The LC-DAD-ESI-MS/MS analysis of gastric and duodenal digested FM (DFM) showed that all compounds remained unchanged at the end of digestion. As proof, a superimposable behavior was observed between FM and DFM in the anti-inflammatory assay carried out on Caco-2 cells. Indeed, it was observed that both FM and DFM decreased the IL-6, IL-8, and nitric oxide (NO) release similarly to the reference anti-inflammatory drug dexamethasone.

Biotransformation of two citrus flavanones by lactic acid bacteria in chemical defined medium.

Microbial processes are being developed to transform flavonoid glycosides to varieties of metabolites with higher bioavailability. The aim of this study was to determine the metabolic activity and survival of five lactic acid bacteria (LAB) stains (L. rhamnosus LRa05, L. casei LC89, L. plantarum N13, L. acidophilus LA85, and L. brevis LB01) in two different citrus flavanone standards (hesperetin-7-O-rutinoside and naringenin-7-O-rutinoside). The enzymatic activity, metabolites, antioxidant activities, and α-glucosidase inhibition property in the two standards were also investigated before and after incubated with LAB. The medium contained standards permitted survival of the five LAB stains. All strains exhibited ß-glucosidase activity. Of the five LAB strains tested, just L. plantarum N13 and L. brevis LB01 have the ability to metabolize hesperetin-7-O-rutinoside, only L. plantarum N13, L. acidophilus LA85, and L. brevis LB01 could metabolize naringenin-7-O-rutinoside, moreover, L. acidophilus LA85l was the strain with the highest biotransformation ratio of naringenin-7-O-rutinoside. L. acidophilus LA85 and L. plantarum N13 can degrade naringenin-7-O-rutinoside into naringenin. L. brevis LB01 can degrade hesperetin-7-O-rutinoside into hesperetin, 3-(4'-hydroxyphenyl)-2-propenoic acid, 3-(3'-hydroxy-4'-methoxyphenyl)hydracrylic acid, and 3-(4'-hydroxyphenyl)propionic acid. Incubation of L. acidophilus LA85 in naringenin-7-O-rutinoside solution supposed no apparent influence in the biological activities that tested. L. acidophilus LA85 may potentially contribute to the bioavailability of citrus flavanones, and to be applied as functional cultures to obtain more bioavailable and bioactive metabolites in food products or in the human gastrointestinal tract.

Development of a LC-HRMS based approach to boost structural annotation of isomeric citrus flavanones.

INTRODUCTION: The structural annotation of target relies on high-resolution mass spectrometry (HRMS) information resulting in dubious identities in most cases. The accurate annotation of isomeric structures is still challenging to be confirmed with significant bottleneck. OBJECTIVE: This study focused on the improvement of structural annotation of candidate structures via four pairs of isomeric flavanone-7-O-diglucosides and their basic flavanone aglycones commonly detected in citrus products. METHOD: An integrated liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) approach merging retention time, accurate mass, tandem mass spectrometry (MS/MS) information (diagnostic ions), ion ratio at selected collision energy was established successfully. RESULTS: Feasibility of this approach was validated confidently in biological samples with relative standard deviation (RSD) of ion ratio range from 3.91 to 12.28%. Differences of fragmentation patterns of citrus flavanones were illustrated reasonably. MS/MS fragments of (S)-hesperetin and (S)-isosakuranetin were complicated and showed typical radical ion [1,2 A - H]•- (m/z 164) in negative ESI mode due to the methoxyl group on B-ring, which showed huge difference with (R)-hesperetin and (R)-isosakuranetin. CONCLUSION: This study integrated multiple levels to boost the confidence of structural annotation relied on LC-HRMS, and provided important values in practice for precise identification of citrus flavanones in biological matrices.

Metabolomics Elucidates Dose-Dependent Molecular Beneficial Effects of Hesperidin Supplementation in Rats Fed an Obesogenic Diet.

Metabolic syndrome (MetS) is a global epidemic concern. Polyphenols are proposed as good candidates for its prevention, although their mechanisms are not fully understood. The gut microbiota seems to play a key role in polyphenol beneficial effects. Here, we assessed the effects of the citrus polyphenol hesperidin combining an untargeted metabolomics approach, which has an inherent potential to elucidate the host-microbiome interplay, with extensive anthropometric and biochemical characterizations and integrating metabolomics results with our previous 16S rRNA bacterial sequencing data. The rats were fed either a standard or an obesogenic cafeteria diet (CAF) for 17 weeks. After nine weeks, rats were supplemented with vehicle; low- (H1), or high- (H2) hesperidin doses. CAF animals developed MetS features. Hesperidin supplementation in CAF rats decreased the total cholesterol, LDL-C, and free fatty acids. The highest hesperidin dose also ameliorated blood pressure, insulin sensitivity, and decreased markers of arterial stiffness and inflammation. Metabolomics revealed an improvement of the lipidomic profile, decreases in circulating amino acids, and lower excretions of inflammation- and oxidative stress-related metabolites. Bacteroidaceae increases in the CAF-H2 group paralleled higher excretions of microbial-derived metabolites. Overall, our results provide detailed insights into the molecular effects of hesperidin on MetS and suggest that it is a promising prebiotic for the treatment of MetS and related conditions.

Therapeutic potential of hesperidin and its aglycone hesperetin: Cell cycle regulation and apoptosis induction in cancer models.

BACKGROUND: The ability of cancer cells to divide without restriction and to escape programmed cell death is a feature of the proliferative state. Citrus flavanones are flavonoids with potential multiple anticancer actions, from antioxidant and chemopreventive, to anti-inflammatory, anti-angiogenic, cytostatic and cytotoxic in different cancer models. PURPOSE: This review aims to summarize the current knowledge on the antiproliferative actions of the citrus flavanones hesperidin (HSD) and hesperetin (HST), with emphasis on cell cycle arrest and apoptosis. METHODS: Cochrane Library, Scopus, Pubmed and Web of Science collection databases were queried for publications reporting antiproliferative effects of HSD and HST in cancer models. RESULTS: HSD and HST have been proven to delay cell proliferation in several cancer models. Depending on the compound, dose and cell line studied, different effects have been reported. Cell cycle arrest associated with cytostatic effects has been reported in cells with increased levels of p53 and also cyclin-dependent kinase inhibitors, as well as decreased levels of specific cyclins and cyclin-dependent kinases. Moreover, apoptotic effects have been found to be associated with altered ratios of pro-/antiapoptotic proteins, caspase activation, c-Jun N-terminal kinase (JNK) pathway activation and caspase-independent pathways. CONCLUSION: Available scientific literature data indicate complex effects, dependent on cell lines and exposure conditions, suggesting that HSD and HST doses need to be optimized according to the cellular and organismal context. The establishment of the main antiproliferative mechanisms is of utmost importance for a possible therapeutic benefit of citrus flavanones in the context of cancer.

The Intestinal Fate of Citrus Flavanones and Their Effects on Gastrointestinal Health.

Citrus flavanones, with hesperidin and naringin as the most abundant representatives, have various beneficial effects, including anti-oxidative and anti-inflammatory activities. Evidence also indicates that they may impact the intestinal microbiome and are metabolized by the microbiota as well, thereby affecting their bioavailability. In this review, we provide an overview on the current evidence on the intestinal fate of hesperidin and naringin, their interaction with the gut microbiota, and their effects on intestinal barrier function and intestinal inflammation. These topics will be discussed as they may contribute to gastrointestinal health in various diseases. Evidence shows that hesperidin and naringin are metabolized by intestinal bacteria, mainly in the (proximal) colon, resulting in the formation of their aglycones hesperetin and naringenin and various smaller phenolics. Studies have also shown that citrus flavanones and their metabolites are able to influence the microbiota composition and activity and exert beneficial effects on intestinal barrier function and gastrointestinal inflammation. Although the exact underlying mechanisms of action are not completely clear and more research in human subjects is needed, evidence so far suggests that citrus flavanones as well as their metabolites have the potential to contribute to improved gastrointestinal function and health.

In Silico Docking and In Vitro Approaches towards BACE1 and Cholinesterases Inhibitory Effect of Citrus Flavanones.

Alzheimer’s disease (AD) is the most prevalent neurodegenerative disease, distinctively characterized by senile plaques, neurofibrillary tangles, and synaptic loss, finally resulting in neuronal death. β-Site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1) and cholinesterases have been identified as therapeutic targets for AD, and the discovery of their inhibitors is of critical importance for developing preventive strategies for AD. To discover natural multi-target compounds possessing BACE1, acetylcholinesterase (AChE), and butyrylcholinesterase (BChE) inhibitory properties, major citrus flavanones including hesperetin, naringenin, and hesperidin were evaluated. In vitro anti-AD activities were performed via BACE1 and cholinesterases inhibition assays, as well as enzyme kinetic predictions. For the design of potential inhibitors of AD-related enzymes, molecular docking analysis was performed. Based on the biological evaluation, hesperidin demonstrated the best inhibitory properties toward BACE1, AChE, and BChE, with IC50 values of 10.02 ± 1.12, 22.80 ± 2.78, and 48.09 ± 0.74 µM, respectively. Kinetic studies revealed that all tested compounds were found to be noncompetitive inhibitors against BACE1 and cholineseterases. In addition, molecular docking studies of these compounds demonstrated negative binding energies for BACE1, AChE, and BChE, indicating high affinity and tight binding capacity for the target enzymes. The present study suggested that the selected citrus flavanones could act together as multiple inhibitors of BACE1, AChE, and BChE, indicating preventive and therapeutic potential against AD.

The acute effects of citrus flavanones on the metabolism of glycogen and monosaccharides in the isolated perfused rat liver.

Citrus flavanones are often linked to their antihyperglycemic properties. This effect may be in part due to the inhibition of hepatic gluconeogenesis through different mechanisms. One of the possible mechanisms appears to be impairment of oxidative phosphorylation, which may also interfere with glycogen metabolism. Based on these facts, the purpose of the present study was to investigate the effects of three citrus flavanones on glycogenolysis in the isolated perfused rat liver. Hesperidin, hesperetin, and naringenin stimulated glycogenolysis and glycolysis from glycogen with concomitant changes in oxygen uptake. At higher concentrations (300 µM), hesperetin and naringenin clearly altered fructose and glucose metabolism, whereas hesperidin exerted little to no effects. In subcellular fractions hesperetin and naringenin inhibited the activity of glucose 6-phosphatase and glucokinase and the mitochondrial respiration linked to ADP phosphorylation. Hesperetin and naringenin also inhibited the transport of glucose into the cell. At a concentration of 300 µM, the glucose influx rate inhibition was 83% and 43% for hesperetin and naringenin, respectively. Hesperidin was the less active among the assayed citrus flavanones, indicating that the rutinoside moiety noticeably decrease the activity of these compounds. The effects on glycogenolysis and fructolysis were mainly consequence of an impairment on mitochondrial energy metabolism. The increased glucose release, due to the higher glycogenolysis, together with glucose transport inhibition is the opposite of what is expected for antihyperglycemic agents.

Citrus flavanones mildly interfere with pituitary-thyroid axis in old-aged male rats.

Citrus flavanones naringenin (NAR) and hesperetin (HES) are potent antioxidants that may contribute to maintenance of health at old age by improving cardiovascular and metabolic status. However, they may also affect thyroid hormone economy. Keeping in mind impaired thyroid function at older age, in this study we tested wheather NAR or HES administration potentiate this decline. NAR or HES were administrated orally (15mg/kg) to male 24-month-old Wistar rats during 4 weeks. Control groups received vehicle, sunflower oil. Qualitative and quantitative immunohistochemical and immunofluorescent expression of specific proteins and stereological analyses of thyroid tissue were performed. Thyroid stimulating hormone (TSH) and total thyroxine (T4) concentrations were measured in serum. Thyroid parenchyma of both flavanone-treated groups was characterized by lower (p<0.05) absolute and relative volume of luminal colloid, accompanied by elevated (p<0.05) relative volume of stroma in comparison with the controls. No hypertrophy or absolute thyroid volume change was detected. Intensity of immunopositive signal for thyroglobulin (Tg) and T4 bound to Tg (T4-Tg) increased (p<0.05) in the colloid of thyroid follicles after both flavanone treatments. Serum TSH increased (p<0.05) after NAR, while T4 remained unchanged after both treatments. In conclusion, NAR elevated serum TSH in old-aged males, thus being more potent than HES in altering pituitary-thyroid axis. However, changes in thyroid structure, namely moderate colloid depletion and higher Tg and T4-Tg protein expressions after both treatments, indicate preserved capacity of the gland to compensate flavanone interfering, and maintain T4 production in old-aged males.

Citrus flavanones naringenin and hesperetin improve antioxidant status and membrane lipid compositions in the liver of old-aged Wistar rats.

This study aimed to investigate effects of citrus flavanones naringenin (NAR) and hesperetin (HES) on liver antioxidant status and membrane phospholipid composition in 24-month-old rats. NAR and HES (15mg/kg) were administrated orally to male Wistar rats, once per day, for 4weeks. Control group received either vehicle (sunflower oil) or remained intact. The results showed decreased (p<0.05) activity of antioxidant enzymes (AOE), specifically catalase (CAT), superoxide dismutase (SOD) 1 and glutathione reductase (GR) in the liver of intact control old-aged rats in comparison to young intact controls. Flavanone administration to old-aged males increased (p<0.05) examined AOE activities in comparison to vehicle-administered animals. Namely, NAR was more potent in comparison to HES regarding the increase (p<0.05) in activities of examined antioxidant enzymes (SOD 1 and 2, glutathione peroxidase-GPx and GR) and the liver glutathione (GSH), while HES elevated (p<0.05) only activity of CAT and GR. Both flavanones significantly decreased (p<0.05) TBARS and improved (p<0.05) membrane phospholipid composition in favor of n-3 PUFA and n-6/n-3 PUFA ratio. Both flavanones did not affect liver histology and reduced (p<0.05) alanine aminotransferase and aspartate aminotransferase levels in serum. The results of this study indicate beneficial potential of citrus flavanones in the old-aged rat liver.

Flavanone-rich citrus beverages counteract the transient decline in postprandial endothelial function in humans: a randomised, controlled, double-masked, cross-over intervention study.

Specific flavonoid-rich foods/beverages are reported to exert positive effects on vascular function; however, data relating to effects in the postprandial state are limited. The present study investigated the postprandial, time-dependent (0-7 h) impact of citrus flavanone intake on vascular function. An acute, randomised, controlled, double-masked, cross-over intervention study was conducted by including middle-aged healthy men (30-65 years, n 28) to assess the impact of flavanone intake (orange juice: 128·9 mg; flavanone-rich orange juice: 272·1 mg; homogenised whole orange: 452·8 mg; isoenergetic control: 0 mg flavanones) on postprandial (double meal delivering a total of 81 g of fat) endothelial function. Endothelial function was assessed by flow-mediated dilatation (FMD) of the brachial artery at 0, 2, 5 and 7 h. Plasma levels of naringenin/hesperetin metabolites (sulphates and glucuronides) and nitric oxide species were also measured. All flavanone interventions were effective at attenuating transient impairments in FMD induced by the double meal (7 h post intake; P<0·05), but no dose-response effects were observed. The effects on FMD coincided with the peak of naringenin/hesperetin metabolites in circulation (7 h) and sustained levels of plasma nitrite. In summary, citrus flavanones are effective at counteracting the negative impact of a sequential double meal on human vascular function, potentially through the actions of flavanone metabolites on nitric oxide.

Molecular mechanisms of citrus flavanones on hepatic gluconeogenesis.

It is well known that hyperglycaemia is the initiating cause of tissue damage associated with type 2 diabetes mellitus and that enhanced hepatic gluconeogenesis may account for the increase in blood glucose levels. The purpose of this work was to investigate the possible actions and mechanisms of three related citrus flavanones, namely hesperidin, hesperetin and naringenin, on hepatic gluconeogenesis and related parameters using isolated perfused rat liver. Hesperetin and naringenin (but not hesperidin) inhibited gluconeogenesis from lactate plus pyruvate, alanine and dihydroxyacetone. The inhibitory effects of these flavanones on gluconeogenesis from lactate and pyruvate (hesperetin IC50 75.6 µM; naringenin IC50 85.5 µM) as well as from alanine were considerably more pronounced than those from dihydroxyacetone. The main cause of gluconeogenesis inhibition is the reduction of pyruvate carboxylation by hesperetin (IC50 134.2 µM) and naringenin (IC50 143.5 µM) via inhibition of pyruvate transport into the mitochondria. Secondary causes are likely inhibition of energy metabolism, diversion of glucose 6-phosphate for glucuronidation reactions and oxidation of NADH by flavanone phenoxyl radicals. The influence of the structural differences between hesperetin and naringenin on their metabolic effects was negligible. Analytical evidence indicated that the presence of a rutinoside moiety in hesperidin noticeably decreases its metabolic effects, confirming that hesperetin and naringenin interact with intracellular enzymes and mitochondrial or cellular membranes better than hesperidin. Thus, the inhibition of the gluconeogenic pathway by citrus flavanones, which was similar to that of the drug metformin, may represent an attractive novel treatment strategy for type 2 diabetes.