Hesperetin protects against rotenone-induced motor disability and neurotoxicity via the regulation of SIRT1/NLRP3 signaling.

Rotenone is a pesticide that causes complex I inhibition and is widely known to induce motor disability and experimental Parkinson's disease (PD) in rodents. Evidence suggests a crucial role for sirtuin/nuclear factor-kappaB/nod-like receptor family, pyrin domain-containing 3 (SIRT1/NFκB/NLRP3) signaling and inflammation in PD and rotenone neurotoxicity. Hesperetin (C16H14O6) is a citrus flavonoid with documented anti-inflammatory activity. We investigated the value of hesperetin in delaying rotenone-induced PD in mice and the possible modulation of inflammatory burden. PD was induced in mice via rotenone injections. Groups were assigned as a vehicle, PD, or PD + hesperetin (50 or 100 mg/kg) and compared for the motor function, protein level (by ELISA), and gene expression (by real-time PCR) of the target proteins, histopathology, and immunohistochemistry for tyrosine hydroxylase enzyme. Hesperetin (50 or 100 mg/kg) alleviated the motor disability and the striatal dopamine level and decreased the expression of NLRP3 and NF-κB but increased SIRT1 expression (p < 0.05). Further, it enhanced the neural viability and significantly decreased neural degeneration in the substantia nigra, hippocampus, and cerebral cortex (p < 0.05). Taken together, we propose that hesperetin mediates its neuroprotective function via alleviating modulation of the SIRT1/NFκB/NLRP3 pathway. Therefore, hesperetin might delay the PD progression.

Hesperetin-loaded chitosan nanoparticles ameliorate hyperglycemia by regulating key enzymes of carbohydrate metabolism in a diabetic rat model.

The study aimed to investigate the potential of hesperetin-loaded chitosan nanoparticles (HSPCNPs) in alleviating hyperglycemia by modulating key enzymes in diabetic rats. Chitosan nanoparticles loaded with hesperetin were prepared using the ionic gelation method and characterized with Electron microscope (SEM), zeta potential, particle size analysis, Fourier-transform infrared (FT-IR), Energy dispersive spectroscopy (EDS) and Encapsulation efficiency and Loading efficiency. To induce diabetes, rats were fed a high-fat beef tallow diet for 28 days, then given a single dose of streptozotocin (STZ) at 35 mg/kg b.w in 0.1 M citrate buffer (pH 4.0). Rats were treated with HSPCNPs at doses of 10, 20, and 40 mg/kg b.w. The analyzed parameters included body weight, food and water intake, plasma glucose and insulin, liver and skeletal muscle glycogen levels, and carbohydrate metabolism. SEM imaging revealed dimensions between 124.2 and 251.6 nm and a mean particle size of 145.0 nm. FT-IR analysis confirmed the presence of functional groups in the chitosan nanoparticles, and the zeta potential was 35.5 mV. HSPCNP 40 mg/kg b.w significantly (p < 0.05) reduced blood glucose levels and glycosylated hemoglobin, improving body weight, food intake, and reducing water intake. In diabetic rats, enzymes for carbohydrate metabolism like fructose 1,6-bisphosphatase, phosphoenolpyruvate carboxykinase, and glucose 6-phosphatase are evaluated in the liver, while glucose 6 phosphate dehydrogenase and hexokinase activity were significantly lower. Additionally, plasma insulin levels increased, indicating enhanced insulin sensitivity. The results show that HSPCNPs at 40 mg/kg b.w. ameliorate hyperglycemia to provide robust protection against diabetic complications and significantly improve metabolic health.

Hesperetin alleviates aflatoxin B1 induced liver toxicity in mice: Modulating lipid peroxidation and ferritin autophagy.

One of the ways Aflatoxin B1 damages the liver is through ferroptosis. Ferroptosis is characterized by the build-up of lipid peroxides and reactive oxygen species (ROS) due to an excess of iron. Dietary supplements have emerged as a promising strategy for treating ferroptosis in the liver. The flavonoid component hesperetin, which is mostly present in citrus fruits, has a number of pharmacological actions, such as those against liver fibrosis, cancer, and hyperglycemia. However, hesperetin's effects and mechanisms against hepatic ferroptosis are still unknown. In this study, 24 male C57BL/6 J mice were randomly assigned to CON, AFB1 (0.45 mg/kg/day), and AFB1+ hesperetin treatment groups (40 mg/kg/day). The results showed that hesperetin improved the structural damage of the mouse liver, down-regulated inflammatory factors (Cxcl1, Cxcl2, CD80, and F4/80), and alleviated liver fibrosis induced by aflatoxin B1. Hesperetin reduced hepatic lipid peroxidation induced by iron accumulation by up-regulating the levels of antioxidant enzymes (GPX4, GSH-Px, CAT, and T-AOC). It is worth noting that hesperetin not only improved lipid peroxidation but also maintained the dynamic balance of iron ions by reducing ferritin autophagy. Mechanistically, hesperetin's ability to regulate ferritin autophagy mostly depends on the PI3K/AKT/mTOR/ULK1 pathway. In AFB1-induced HepG2 cells, the addition of PI3K inhibitor (LY294002) and AKT inhibitor (Miransertib) confirmed that hesperetin regulated the PI3K/AKT/mTOR/ULK1 pathway to inhibit ferritin autophagy and reduced the degradation of ferritin in lysosomes. In summary, our results suggest that hesperetin not only regulates the antioxidant system but also inhibits AFB1-induced ferritin hyperautophagy, thereby reducing the accumulation of iron ions to mitigate lipid peroxidation. This work provides a fresh perspective on the mechanism behind hesperetin and AFB1-induced liver damage in mice.

Impact of Liposomal Hesperetin in Broilers: Prospects for Improving Performance, Antioxidant Potential, Immunity, and Resistance against Listeria monocytogenes.

Liposomal encapsulated phytogenics, such as liposomal hesperetin, are considered novel substitutes for antibiotics in the broilers industry owing to their improved nutritional and therapeutic properties. Therefore, our key goal was to investigate liposomal hesperetin impact on broilers' growth performance, health, antioxidant status, tight junction proteins (TJP), and resistance against Listeria monocytogenes. Four broilers' groups were fed 0, 150, 250, and 400 mg/kg of liposomal hesperetin-supplemented diets and experimentally infected with L. monocytogenes strain. Herein, liposomal hesperetin, especially at higher concentrations, augmented broilers FCR with upregulation of genes encoding TJP (occludin, JAM-2, MUC-2), and antioxidant attributes (GPX-1, SOD-1, CAT, HO-1, NQO1, COX2), which reflect enhancing health and welfare of broilers. Muscle antioxidant biomarkers were enhanced; meanwhile, muscle MDA, ROS, and H2O2 levels were reduced in response to 400 mg/kg of liposomal hesperetin. Liposomal hesperetin fortification reduced L. monocytogenes loads and its virulence-related genes expression levels (flaA, hlyA, and ami). Remarkably, histopathological alterations in intestinal and brain tissues of L. monocytogenes infected broilers were restored post-inclusion at higher levels liposomal hesperetin, which reflects increasing the bird's resistance to L. monocytogenes infection. Transcription levels of genes encoding cytokines/chemokines (MyD88, AVBD6, CCL20, IL-1ß, IL-18), and autophagy (Bcl-2, LC3, AMPK, AKT, CHOP, Bip, p62, XBP1) were ameliorated following dietary liposomal hesperetin fortification, which suggests enhancing the birds' immunity and health. Collectively, our research recommends liposomal hesperetin application in broilers` diets owing to its promoting impact on growth performance, antioxidant status, immunity, health, and welfare besides its antibacterial, and antivirulence characteristics to fight against L. monocytogenes.

Hesperetin regulates PI3K/Akt and mTOR pathways to exhibit its antiproliferative effect against colon cancer cells.

Hesperetin, a citrus flavonoid, has been a widely studied anticancer agent against many types of cancers, but the exact mechanism of efficacy is still unrevealed. Therefore, this study has attempted to delineate the mechanical aspect of hesperetin's anticancer efficacy against colon cancer using immunoblotting, scanning, and transmission electron microscopic studies. The treatment with hesperetin (25 and 50 µM) has significantly (p < 0.0001) curbed down the proliferation and cell viability of HCT-15 cells in a concentration as well as time dependent manner. Hesperetin was able to achieve this through the induction of caspase-dependent apoptosis. Moreover, hesperetin effectively inhibited phosphorylation of Akt with a parallel increase in PTEN expression thereby inhibiting the PI3K signaling axis, which contributes to the suppression of proliferation. In addition, hesperetin enhanced autophagy through dephosphorylating mTOR, one of the downstream targets of Akt with simultaneous acceleration in Beclin-1 and LC3-II expression levels. Interestingly, hesperetin enhanced the effects of Akt inhibitor LY294002 and mTOR inhibitor rapamycin. This study documented the potential of hesperetin to induce apoptosis through simultaneous acceleration over the autophagic process in colon cancer cells. Thus, hesperetin played a beneficial therapeutic role in preventing colon carcinoma growth by regulating the Akt and mTOR signaling axis.

Hesperetin-7-O-rhamnoglucoside ameliorates dichlorvos-facilitated cardiotoxicity in rats by counteracting ionoregulatory, ion pumps, redox, and lipid homeostasis disruptions.

The contamination of edible agricultural goods with pesticides, including dichlorvos (DVDP), poses a substantial public health risk, promoting severe morbidity and mortality, especially in developing countries. It has been shown that hesperidin (hesperetin-7-O-rhamnoglucoside or Hes-7-RGlc) preserves cytomembrane, redox, and lipid homeostasis; unfortunately, its function on dichlorvos-incited heart damage has not been investigated. This work explored the ameliorative influence of Hes-7-RGlc on DVDP-activated cardiotoxicity. For this end, forty-two rats were randomly appropriated into seven groups (6 rats/group): Control, DVDP alone (8 mg.kg⁻¹day⁻¹), DVDP supplied with either Hes-7-RGlc (50 and 100 mg.kg⁻¹day⁻¹) or the reference medication atropine (0.2 mg.kg⁻¹day⁻¹), and Hes-7-RGlc alone (50 and 10 mg.kg⁻¹day⁻¹) were the seven groups investigated. DVDP was administered orally for seven days, followed by fourteen days of Hes-7-RGlc therapy. Then the rats were euthanized, and their blood and hearts were removed. Hes-7-RGlc chemotherapy substantially (p<0.05) restored DVDP-elicited dynamics in plasma and cardiac/myocardium creatine kinase isoenzyme (CK-MB), major lipids (cholesterol, triacylglycerol, and phospholipids), electrolytes (Na⁺, K⁺, Ca²âº, Mg²âº, Cl⁻), and total protein. Hes-7-RGlc remedy decidedly (p<0.05) abolished DDVP-stimulated amplification in the cardiac concentration of H2O2, NO and malondialdehyde; annulled DVDP-educed decreases in heart GSH levels, activities of GST, SOD, catalase, and glutathione peroxidase, ion transporters (Na⁺/K⁺-ATPase and Ca²âº/Mg²âº-ATPase), ALT, AST, ALP, and LDH-1. Collectively, Hes-7-RGlc can be advocated as a natural supplementary candidate and blocker of DVDP-provoked heart deficits via its capacity to reverse disruptions of electrolytes, ion pumps, redox status, and lipid homeostasis.

Hesperetin Alleviated Experimental Colitis via Regulating Ferroptosis and Gut Microbiota.

Hesperetin (HT) is a type of citrus flavonoid with various pharmacological activities, including anti-tumor, anti-inflammation, antioxidant, and neuroprotective properties. However, the role and mechanism of HT in ulcerative colitis (UC) have been rarely studied. Our study aimed to uncover the beneficial effects of HT and its detailed mechanism in UC. Experimental colitis was induced by 2.5% dextran sodium sulfate (DSS) for seven days. HT ameliorated DSS-induced colitis in mice, showing marked improvement in weight loss, colon length, colonic pathological severity, and the levels of TNFα and IL6 in serum. A combination of informatics, network pharmacology, and molecular docking identified eight key targets and multi-pathways influenced by HT in UC. As a highlight, the experimental validation demonstrated that PTGS2, a marker of ferroptosis, along with other indicators of ferroptosis (such as ACSL4, Gpx4, and lipid peroxidation), were regulated by HT in vivo and in vitro. Additionally, the supplement of HT increased the diversity of gut microbiota, decreased the relative abundance of Proteobacteria and Gammaproteobacteria, and restored beneficial bacteria (Lachnospiraceae_NK4A136_group and Prevotellaceae_UCG-001). In conclusion, HT is an effective nutritional supplement against experimental colitis by suppressing ferroptosis and modulating gut microbiota.

Mechanistic study on vasodilatory and antihypertensive effects of hesperetin: ex vivo and in vivo approaches.

Hesperetin is one of the prominent flavonoids found in citrus fruit. Several research studies have reported that hesperetin can promote vasodilation in vascular tissue by increasing the level of nitric oxide and cyclic nucleotides. However, these may not be the only pathway for hesperetin to exert its vasodilatory effect. In addition to vasodilation, hesperetin has been found to carry an antihypertensive effect through intraperitoneal injection, although no study has comprehensively investigated the antihypertensive effect of hesperetin through oral administration. Therefore, this study aimed to determine the possible mechanism pathways involved in hesperetin-induced vasodilation and investigated its antihypertensive effects on hypertensive rats' model via oral administration. The ex vivo experimental findings showed that the NO/sGC/cGMP signalling pathway was involved in hesperetin-mediated vasodilation. Moreover, hesperetin activated the AC/cAMP/PKA pathway through PGI2 and activated the ß2-adrenergic receptor. Hesperetin can act as a voltage-gated potassium channel (KV) and ATP-sensitive potassium channel (KATP) opener. The intracellular calcium in vascular smooth muscle was reduced by hesperetin through blocking the voltage-operated calcium channels (VOCC) and inositol triphosphate receptor (IP3R). In the in vivo assessment, hesperetin shows a significant decrease in Spontaneously Hypertensive rats' blood pressure following 21 days of oral treatment. The sub-chronic toxicity assessment demonstrated that hesperetin exhibited no deleterious effects on the body weights, clinical biochemistry and haematological profile of Sprague-Dawley rats. This study implies that hesperetin holds promise as a potential medication for hypertension treatment, devoid of undesirable side effects.

Butyrate improves recovery from experimental necrotizing enterocolitis by metabolite hesperetin through potential inhibition the PI3K-Akt pathway.

Necrotizing enterocolitis (NEC) is one of the most common and serious intestinal illnesses in newborns and seriously affects their long-term prognosis and survival. Butyrate is a short-chain fatty acid that can relieve intestinal inflammation, but its mechanism of action is unclear. Results from an in vivo neonatal rat model has shown that butyrate caused an improved recovery from NEC. These protective effects were associated with the metabolite of hesperetin, as determined by metabolomics and molecular biological analysis. Furthermore, transcriptomics combined with inhibitor assays were used to investigate the mechanism of action of hesperetin in an in vitro NEC model (IEC-6 cells exposed to LPS) to further investigate the mechanism by which butyrate attenuates NEC. The transcriptomics analysis showed that the PI3K-Akt signaling pathway was involved in the anti-NEC effect of hesperitin. Subsequently, the results using an inhibitor of PI3K (LY294002) indicated that the suppression could be explained by the hesperetin-induced expression of tight junction (TJ) proteins by potentially blocking the PI3K-Akt signaling pathway. In summary, the present study demonstrated that butyrate could improve recovery from NEC with a hesperetin metabolite, causing potential inhibition of the phosphorylation of the PI3K-Akt signaling pathway, resulting in the increased expression of TJ proteins. These findings reveal a potential new therapeutic pathway for the treatment of NEC.

Formulation of a Novel Hesperetin-Loaded Nanoemulsion and Its Promising Effect on Osteogenesis.

Alternative therapies associating natural products and nanobiotechnology show new perspectives on controlled drug release. In this context, nanoemulsions (NEs) present promising results for their structural design and properties. Hesperetin (HT), a flavonoid mainly found in citrus fruits, presents highlighted bone benefits. In this context, we developed a hesperetin-loaded nanoemulsion (HT-NE) by sonication method and characterized it by dynamic light scattering, analyzing its encapsulation efficiency, and cumulative release. The biocompatibility in human osteoblasts Saos-2-like was evaluated by the cytotoxicity assay and IC50. Then, the effects of the HT-NE on osteogenesis were evaluated by the cellular proliferation, calcium nodule formation, bone regulators gene expression, collagen quantification, and alkaline phosphatase activity. The results showed that the formulation presented ideal values of droplet size, polydispersity index, and zeta potential, and the encapsulation efficiency was 74.07 ± 5.33%, showing a gradual and controlled release. Finally, HT-NE was shown to be biocompatible and increased cellular proliferation, and calcium nodule formation, regulated the expression of Runx2, ALPL, and TGF-ß genes, and increased the collagen formation and alkaline phosphatase activity. Therefore, the formulation of this NE encapsulated the HT appropriately, allowing the increasing of its effects on mechanisms to improve or accelerate the osteogenesis process.

In Vitro Biological Activities of Hesperidin-Related Compounds with Different Solubility.

The biological activities of hesperidin-related compounds, such as hesperetin laurate (HTL), hesperetin (HT), hesperidin (HD), and hesperidin glucoside (HDG), were investigated in vitro. The compounds showed different hydrophobicities, and the octanol-water partition coefficient log P were 7.28 ± 0.06 for HTL, 2.59 ± 0.04 for HT, 2.13 ± 0.03 for HD, and -3.45 ± 0.06 for HDG, respectively. In the DPPH assay and ß-carotene bleaching assay to determine antioxidant capacity, all compounds tested showed antioxidant activity in a concentration-dependent manner, although to varying degrees. HTL and HT showed similarly high activities compared to HD or HDG. HD and HDG did not show a significant difference despite the difference in solubility between the two. Cytotoxicity was high; in the order of hydrophobicity-HTL > HT > HD > HDL in keratinocyte HaCaT cells. All compounds tested showed reducing effects on cellular inflammatory mediators and cytokines induced by UV irradiation. However, HTL and HT effectively reduced nitric oxide (NO), tumor necrosis factor α (TNF-α), and interleukin-6 (IL-6) levels compared to HD and HDG. The inhibitory effects of hesperidin-related compounds on skin-resident microorganisms were evaluated by measuring minimum inhibitory concentration (MIC). HTL showed the highest inhibitory effects against Staphylococcus aureus, Cutibacterium acnes, Candida albicans, and Malassezia furfur, followed by HT, while HD and HDF showed little effect. In conclusion, the hydrophobicity of hesperidin-related compounds was estimated to be important for biological activity in vitro, as was the presence or absence of the sugar moiety.

Experimental and Computational Studies on the Interaction of DNA with Hesperetin Schiff Base CuII Complexes.

The interactions with calf thymus DNA (CT-DNA) of three Schiff bases formed by the condensation of hesperetin with benzohydrazide (HHSB or L1H3), isoniazid (HIN or L2H3), or thiosemicarbazide (HTSC or L3H3) and their CuII complexes (CuHHSB, CuHIN, and CuHTSC with the general formula [CuLnH2(AcO)]) were evaluated in aqueous solution both experimentally and theoretically. UV-Vis studies indicate that the ligands and complexes exhibit hypochromism, which suggests helical ordering in the DNA helix. The intrinsic binding constants (Kb) of the Cu compounds with CT-DNA, in the range (2.3-9.2) × 106, from CuHTSC to CuHHSB, were higher than other copper-based potential drugs, suggesting that π-π stacking interaction due to the presence of the aromatic rings favors the binding. Thiazole orange (TO) assays confirmed that ligands and Cu complexes displace TO from the DNA binding site, quenching the fluorescence emission. DFT calculations allow for an assessment of the equilibrium between [Cu(LnH2)(AcO)] and [Cu(LnH2)(H2O)]+, the tautomer that binds CuII, amido (am) and not imido (im), and the coordination mode of HTSC (O-, N, S), instead of (O-, N, NH2). The docking studies indicate that the intercalative is preferred over the minor groove binding to CT-DNA with the order [Cu(L1H2am)(AcO)] > [Cu(L2H2am)(AcO)] ≈ TO ≈ L1H3 > [Cu(L3H2am)(AcO)], in line with the experimental Kb constants, obtained from the UV-Vis spectroscopy. Moreover, dockings predict that the binding strength of [Cu(L1H2am)(AcO)] is larger than [Cu(L1H2am)(H2O)]+. Overall, the results suggest that when different enantiomers, tautomers, and donor sets are possible for a metal complex, a computational approach should be recommended to predict the type and strength of binding to DNA and, in general, to macromolecules.

Sweeteners Show a Plasticizing Effect on PVP K30-A Solution for the Hot-Melt Extrusion of Fixed-Dose Amorphous Curcumin-Hesperetin Solid Dispersions.

The co-administration of curcumin and hesperetin might be beneficial in terms of neuroprotective activity; therefore, in this study, we attempted to develop a fixed-dose formulation comprising these two compounds in an amorphous state. The aim of obtaining an amorphous state was to overcome the limitations of the low solubility of the active compounds. First, we assessed the possibility of using popular sweeteners (erythritol, xylitol, and sorbitol) as plasticizers to reduce the glass transition temperature of PVP K30 to prepare the polymer-excipient blends, which allowed the preparation of amorphous solid dispersions via hot-melt extrusion at a temperature below the original glass transition of PVP K30. Erythritol proved to be the superior plasticizer. Then, we focused on the development of fixed-dose amorphous solid dispersions of curcumin and hesperetin. Powder X-ray diffraction and thermal analysis confirmed the amorphous character of dispersions, whereas infrared spectroscopy helped to assess the presence of intermolecular interactions. The amorphous state of the produced dispersions was maintained for 6 months, as shown in a stability study. Pharmaceutical parameters such as dissolution rate, solubility, and in vitro permeability through artificial membranes were evaluated. The best improvement in these features was noted for the dispersion, which contained 15% of the total content of the active compounds with erythritol used as the plasticizer.

Elevated PINK1/Parkin-Dependent Mitophagy and Boosted Mitochondrial Function Mediate Protection of HepG2 Cells from Excess Palmitic Acid by Hesperetin.

Deregulation of mitochondrial functions in hepatocytes contributes to many liver diseases, such as nonalcoholic fatty liver disease (NAFLD). Lately, it was referred to as MAFLD (metabolism-associated fatty liver disease). Hesperetin (Hst), a bioactive flavonoid constituent of citrus fruit, has been proven to attenuate NAFLD. However, a potential connection between its preventive activities and the modulation of mitochondrial functions remains unclear. Here, our results showed that Hst alleviates palmitic acid (PA)-triggered NLRP3 inflammasome activation and cell death by inhibition of mitochondrial impairment in HepG2 cells. Hst reinstates fatty acid oxidation (FAO) rates measured by seahorse extracellular flux analyzer and intracellular acetyl-CoA levels as well as intracellular tricarboxylic acid cycle metabolites levels including NADH and FADH2 reduced by PA exposure. In addition, Hst protects HepG2 cells against PA-induced abnormal energetic profile, ATP generation reduction, overproduction of mitochondrial reactive oxygen species, and collapsed mitochondrial membrane potential. Furthermore, Hst improves the protein expression involved in PINK1/Parkin-mediated mitophagy. Our results demonstrate that it restores PA-impaired mitochondrial function and sustains cellular homeostasis due to the elevation of PINK1/Parkin-mediated mitophagy and the subsequent disposal of dysfunctional mitochondria. These results provide therapeutic potential for Hst utilization as an effective intervention against fatty liver disease.

Novel Catechol O-methyltransferases from Lentinula edodes Catalyze the Generation of Taste-Active Flavonoids.

Due to the increasing demand for natural food ingredients, including taste-active compounds, enzyme-catalyzed conversions of natural substrates, such as flavonoids, are promising tools to align with the principles of Green Chemistry. In this study, a novel O-methyltransferase activity was identified in the mycelium of Lentinula edodes, which was successfully applied to generate the taste-active flavonoids hesperetin, hesperetin dihydrochalcone, homoeriodictyol, and homoeriodictyol dihydrochalcone. Furthermore, the mycelium-mediated OMT activity allowed for the conversion of various catecholic substrates, yielding their respective (iso-)vanilloids, while monohydroxylated compounds were not converted. By means of a bottom-up proteomics approach, three putative O-methyltransferases were identified, and subsequently, synthetic, codon-optimized genes were heterologously expressed in Escherichia coli. The purified enzymes confirmed the biocatalytic O-methylation activity against targeted flavonoids containing catechol motifs.

A New Hyaluronic Emulgel of Hesperetin for Topical Application-An In Vitro Evaluation.

The present study aimed to formulate and characterize a hesperetin formulation to achieve adequate deposition and retention of hesperetin in the epidermis as a target for some cosmetic/dermatological actions. To derive the final emulgel, various formulations incorporating different proportions of Polysorbate 80 and hyaluronic acid underwent testing through a Box-Behnken experimental design. Nine formulations were created until the targeted emulgel properties were achieved. This systematic approach, following the principles of a design of experiment (DoE) methodology, adheres to a quality-by-design (QbD) paradigm, ensuring a robust and purposeful formulation and highlighting the commitment to a quality-driven design approach. The emulsions were developed using the phase inversion method, optimizing the emulgel with the incorporation of hyaluronic acid. Physically stable optimized emulgels were evaluated for their globule size, surface charge, viscosity, pH, electrical conductivity, and hesperetin content. These assays, along with the temperature swing test, were used to select the optimal formulation. It was characterized by a droplet size, d[4,3], of 4.02 µm, a Z-potential of -27.8 mV, an O/W sign, a pH of 5.2, and a creamy texture and proved to be stable for at least 2 months at room temperature. Additionally, in vitro release kinetics from the selected emulgel exhibited a sustained release profile of hesperetin. Skin assays revealed adequate retention of hesperetin in the human epidermis with minimum permeation. Altogether, these results corroborate the promising future of the proposed emulgel in cosmetic or dermatological use on healthy or diseased skin.

Comparative evaluation of hesperetin-loaded graphene oxide nanosheets (Hsp-GO) as a drug delivery system for colon cancer: synthesis and anticancer efficiency assessment.

BACKGROUND: Graphene oxide nanosheets (GONS) are recognized for their role in enhancing drug delivery and effectiveness in cancer treatment. With colon cancer being a prevalent global issue and the significant side effects associated with chemotherapy, the primary treatment for colon cancer alongside surgery, there is a critical need for novel therapeutic strategies to support patients in combating this disease. Hesperetin (HSP), a natural compound found in specific fruits, exhibits anti-cancer properties. The aim of this study is to investigate the effect of GONS on the LS174t colon cancer cell line. METHODS: In this study, an anti-cancer nano-drug was synthesized by creating a hesperetin-graphene oxide nanocomposite (Hsp-GO), which was subsequently evaluated for its efficacy through in vitro cell toxicity assays. Three systems were investigated: HSP, GONS, and HSP-loaded GONS, to determine their cytotoxic and pro-apoptotic impacts on the LS174t colon cancer cell line, along with assessing the expression of BAX and BCL2. The morphology and properties of both GO and Hsp-GO were examined using scanning electron microscopy (SEM), X-ray diffraction, and Fourier transform infrared spectroscopy (FTIR). RESULTS: The Hsp-GO nanocomposite displayed potent cytotoxic and pro-apoptotic effects on LS174t colon cancer cells, outperforming individual treatments with HSP or GONS. Cell viability assays showed a significant decrease in cell viability with Hsp-GO treatment. Analysis of BAX and BCL2 expression revealed elevated BAX and reduced BCL2 levels in Hsp-GO treated cells, indicating enhanced apoptotic activity. Morphological analysis confirmed successful Hsp-GO synthesis, while structural integrity was supported by X-ray diffraction and FTIR analyses. CONCLUSIONS: These study highlight the potential of Hsp-GO as a promising anti-cancer nano-drug for colon cancer therapy.

Anti-Parkinson potential of hesperetin nanoparticles: in vivo and in silico investigations.

Parkinson's disease (PD) is characterised by the gradual demise of dopaminergic neurons. In recent years, there has been significant interest in herbal treatments. In this study, hesperetin nanoparticles (HTN) were developed and compared their anti-PD potential with hesperetin (HT) on rotenone induced PD rats. Molecular docking was also performed to evaluate the binding affinity of hesperetin on pathological protein, i.e. D2 dopamine receptors (DR2), using Auto Dock Vina tools. The results showed a higher binding relationship of HTN on dopamine receptors (-7.2 kcal/mol) compared to L-dopa (-6.4 kcal/mol), supporting their potential as drug candidates for PD therapy. HTN was effectively synthesised using the fabrication technique and characterised by zeta potential and SEM analysis. HTN had favourable characteristics, including a size of 249.8 ± 14.9 nm and a Z-potential of -32.9 mV. After being administered orally, HTN demonstrated a notable anti-Parkinsonian effects, indicated by the significant improvement in motor function as assessed by the rota rod test (p < .001***), pole test (p < .001***), stair test (p < .01**), wood walk test (p < .01**) and an increase in substantia nigra (SN) antioxidant levels, CAT (p < .001***), SOD (p < .001***), GSH (p < .01**). Additionally, HTN led to increased dopamine levels (p < .01**) and a decrease in the oxidant system, MDA levels (p < .01**). Furthermore, histopathological examination revealed decreased SN neuronal necrosis in diseased animals treated with HTN compared to those treated with HT in a rat model of Parkinson's disease. Therefore, HTN can be regarded as a viable platform for efficient therapy of PD.

Hesperetin ameliorates spinal cord injury in rats through suppressing apoptosis, oxidative stress and inflammatory response.

Spinal cord injury (SCI), a fatal condition, is characterized by progressive tissue degradation and extreme functional deficits with limited treatment options. Hesperetin, a natural flavonoid with potent antioxidant, antiapoptotic and anti-inflammatory properties, has yet to be systematically investigated for its therapeutic effects on neurological damage in rat models of SCI. In this study, rats were given oral hesperetin once daily for 28 days, and their locomotion and histopathological changes were assessed. The findings demonstrated that hesperetin alleviates neurological damage caused by SCI. The observed behavioral improvement could be due to an increase in the survival rate of neurons and oligodendrocytes. This improvement further boosted the ability to repair tissue and form myelin after SCI, ultimately resulting in better neurological outcomes. Furthermore, the present study revealed that hesperetin possesses potent antioxidant capabilities in the context of SCI, reducing the levels of harmful oxygen free radicals and increasing the activity of antioxidant enzymes. Additionally, hesperetin markedly inhibited injury-induced apoptosis, as assessed by caspase-3 immunofluorescence staining and the expression level of caspase-3, indicating the ability of hesperetin to prevent cell death after SCI. Finally, after SCI, hesperetin treatment effectively reduced the expression of inflammatory factors, including IL-1ß, TNFα, and NF-kB, demonstrating the anti-inflammatory effect of hesperetin. Together, our results suggest that hesperetin should be considered a valuable therapeutic aid following SCI, as its positive effects on the nervous system, including antioxidant, anti-inflammatory and antiapoptotic effects, may be crucial mechanisms through which hesperetin exerts neuroprotective effects against SCI.

Cellular electrophysiological effects of the citrus flavonoid hesperetin in dog and rabbit cardiac ventricular preparations.

Recent experimental data shows that hesperetin, a citrus flavonoid, affects potassium channels and can prolong the QTc interval in humans. Therefore, in the present study we investigated the effects of hesperetin on various transmembrane ionic currents and on ventricular action potentials. Transmembrane current measurements and action potential recordings were performed by patch-clamp and the conventional microelectrode techniques in dog and rabbit ventricular preparations. At 10 µM concentration hesperetin did not, however, at 30 µM significantly decreased the amplitude of the IK1, Ito, IKr potassium currents. Hesperetin at 3-30 µM significantly and in a concentration-dependent manner reduced the amplitude of the IKs current. The drug significantly decreased the amplitudes of the INaL and ICaL currents at 30 µM. Hesperetin (10 and 30 µM) did not change the action potential duration in normal preparations, however, in preparations where the repolarization reserve had been previously attenuated by 100 nM dofetilide and 1 µg/ml veratrine, caused a moderate but significant prolongation of repolarization. These results suggest that hesperetin at close to relevant concentrations inhibits the IKs outward potassium current and thereby reduces repolarization reserve. This effect in certain specific situations may prolong the QT interval and consequently may enhance proarrhythmic risk.