In vitro evaluation of PLGA loaded hesperidin on colorectal cancer cell lines: an insight into nano delivery system.

BACKGROUND: Colorectal cancer is a common disease worldwide with non-specific symptoms such as blood in the stool, bowel movements, weight loss and fatigue. Chemotherapy drugs can cause side effects such as nausea, vomiting and a weakened immune system. The use of antioxidants such as hesperidin could reduce the side effects, but its low bioavailability is a major problem. In this research, we aimed to explore the drug delivery and efficiency of this antioxidant on the HCT116 colorectal cancer cell line by loading hesperidin into PLGA nanoparticles. MATERIALS AND METHODS: Hesperidin loaded PLGA nanoparticles were produced by single emulsion evaporation method. The physicochemical properties of the synthesized hesperidin-loaded nanoparticles were determined using SEM, AFM, FT-IR, DLS and UV-Vis. Subsequently, the effect of the PLGA loaded hesperidin nanoparticles on the HCT116 cell line after 48 h was investigated by MTT assay at three different concentrations of the nanoparticles. RESULT: The study showed that 90% of hesperidin were loaded in PLGA nanoparticles by UV-Vis spectrophotometry and FT-IR spectrum. The nanoparticles were found to be spherical and uniform with a hydrodynamic diameter of 76.2 nm in water. The release rate of the drug was about 93% after 144 h. The lowest percentage of cell viability of cancer cells was observed at a concentration of 10 µg/ml of PLGA nanoparticles loaded with hesperidin. CONCLUSION: The results indicate that PLGA nanoparticles loaded with hesperidin effectively reduce the survival rate of HCT116 colorectal cancer cells. However, further studies are needed to determine the appropriate therapeutic dosage and to conduct animal and clinical studies.

Enhanced Therapeutic Efficacy Against Melanoma through Exosomal Delivery of Hesperidin.

Melanoma, characterized as the most aggressive and metastatic form of skin cancer, currently has limited treatment options, predominantly chemotherapy and radiation therapy. However, the drawbacks associated with parenterally administered chemotherapy underscore the urgent need for alternative compounds to combat melanoma effectively. Hesperidin (HES), a flavonoid present in various citrus fruits, exhibits promising anticancer activity. Nevertheless, the clinical utility of HES is hindered by challenges such as poor water solubility, a short half-life, and low oral bioavailability. In response to these limitations, we introduced a novel approach by formulating HES-loaded exosomes (Exo-HES). Isolation of exosomes was achieved through the ultracentrifugation method, and HES was efficiently loaded using the sonication method. The resulting formulations displayed a desirable particle size (∼106 nm) and exhibited a spherical morphology, as confirmed by scanning electron and atomic force microscopy. In vitro studies conducted on B16F10 cell lines demonstrated higher cytotoxicity of Exo-HES compared to free HES, supported by enhanced cellular uptake validated through coumarin-6-loaded exosomes. This superior cytotoxicity was further evidenced by DNA fragmentation, increased generation of free radicals (ROS), loss of mitochondrial membrane potential, and effective inhibition of colony formation. The antimetastatic properties of Exo-HES were confirmed through wound healing and transwell migration assays. Oral pharmacokinetics studies revealed a remarkable increase of approximately 2.5 times in oral bioavailability and half-life of HES when loaded into exosomes. Subsequent in vivo experiments utilizing a B16F10-induced melanoma model in Swiss mice established that Exo-HES exhibited superior anticancer activity compared to HES after oral administration. Importantly, no biochemical, hematological, or histological toxicities were observed in tumor-bearing mice treated with Exo-HES. These findings suggest that exosomes loaded with HES represent a promising nanocarrier strategy to enhance the therapeutic effectiveness of hesperidin in melanoma treatment.

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.

Thermoresponsive lipids engineered magnetic nanoparticles for spatiotemporal delivery of hesperidin to inflammatory sites in animal model.

Thermoresponsive nanoparticles are exploited as drug-delivery vehicles that release their payload upon increment in temperature. We prepared and characterized thermoresponsive lipid-anchored folic acid engineered magnetic nanoparticles (LP-HP-FANPs) that combine receptor-based targeting and thermoresponsive sustained release of hesperidin (HP) in response to endogenous inflammation site temperature. The progressive surface engineering of NPs was validated by FTIR analysis. Our LP-HP-FANPs had a particle size of 100.5 ± 1.76 nm and a zeta potential of 14.6 ± 2.65 mV. The HP encapsulation effectiveness of LP-HP-FANPs is around 91 ± 0.78%. AFM scans indicated that our modified nanoparticles were spherical. LP-HP-FANPs exhibit increased drug release (85.8% at pH 4.0, 50.9% at pH 7.0) at 40 °C. Animal studies showed no toxicity from nanoparticles. Compared to conventional drugs and HP, LP-HP-FANPs effectively decreased paw edema, cytokine levels, and total cell recruitment in thioglycollate-induced peritonitis (p < 0.05). LP-HP-FANPs substantially decreased cytokines compared to HP, HP-FA-NPs, and the standard medication (p < 0.05, p < 0.01, and p < 0.001). These findings imply that the synthesized HP-loaded formulation (LP-HP-FANPs) may be a potential anti-inflammatory formulation for clinical development.

Solid lipid nanoparticles (SLNs), the potential novel vehicle for enhanced in vivo efficacy of hesperidin as an anti-inflammatory agent.

Hesperidin (C28H34O15), a flavanone glycoside abundantly present in citrus fruits, has proven therapeutic effects including anti-inflammatory activities. Herein, we report a novel formulation of HESP loaded solid lipid nanoparticles (SLNs) using hot homogenization and ultrasound to improve the poor solubility and bioavailability. In the present study, the formulation was developed and optimized by response surface method and then characterized by scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy (FT-IR), and dynamic light scattering (DLS). Encapsulation efficiency was determined and the anti-inflammatory effect was assessed through in vivo ear edema inflammation model. According to the electron microscopy results, the product has a spherical shape. The optimized parameters produced small size (179.8 ± 3.6 nm) HESP-SLNs with high encapsulation efficiency (93.0 ± 3.8 %). The outcomes exhibited that encapsulation in SLNs carriers improves the anti-inflammatory potential of HESP.

Improved bioavailability and anticancer efficacy of Hesperetin on breast cancer via a self-assembled rebaudioside A nanomicelles system.

Hesperetin (HSP) has excellent biological activities with poor water solubility which limits its clinical development. In this study, we successfully prepared a novel, self-assembled micelle based on Rebaudioside A (RA) for oral delivery of HSP with improved bioavailability and therapeutic effects. We found that RA and HSP could be formylated into nanomicelles with particle sizes of 4.541 nm ± 0.048 nm. HSP was readily encapsulated into RA micelles and this improved its water solubility (to 12.74 mg/mL ± 0.28 mg/mL). The MTT results showed that RA-HSP enhanced the cytotoxicity, the clonal formation inhibitory activity, and cell migration inhibitory activity of HSP in human breast cancer MDA-MB-231 cells. The mechanism results showed that RA-HSP induced cell apoptosis by inducing the production of reactive oxygen species (ROS), destroying the mitochondrial membrane potential (MMP), and inhibiting the PI3K/Akt signaling pathway. Moreover, RA-HSP enhanced the anticancer activity, increased the oral bioavailability and tissue distribution of HSP in vivo. Moreover, the mechanism studies in vivo found that HSP inhibited PI3K/Akt signaling pathway with low side effects. These findings indicate that RA micelle formulations have great potential in oral drug delivery systems for the delivery of hydrophobic drugs.

Hesperidin opposes the negative impact of cyclophosphamide on mice kidneys.

The present investigation examined the prospective nephroprotective effect of hesperidin (HSN) in mice challenged with a single i.p. injection of cyclophosphamide (CPE) at a dose of 200 mg/kg. HSN (100 and 200 mg/kg/day, p.o.) was given for 10 days, starting 5 days prior to CPE administration. HSN significantly reduced the CPE-induced increments of serum creatinine and cystatin C. HSN also significantly reduced malondialdehyde, nitric oxide, Bax/Bcl-2 ratio, and caspase-3, and significantly raised total antioxidant capacity, and interleukin-10/tumor necrosis factor-α ratio in kidneys of mice received CPE. In addition, HSN significantly prevented the histopathological injury, and kidney injury molecule-1 expression in kidneys of mice given CPE. It was concluded that HSN guarded against nephrotoxic effect of CPE in mice by tackling oxidative/nitrative stress, inflammation, and apoptosis.

Solid self-microemulsifying nutraceutical delivery system for hesperidin using quality by design: assessment of biopharmaceutical attributes and shelf-life.

AIM: The present study endeavours to develop a solid self-microemulsifying nutraceutical drug delivery system for hesperidin (HES) using quality by design (QbD) to improve its biopharmaceutical attributes. METHODS: A 32 full factorial design was employed to study the influence of factors on selected responses. Risk assessment was performed by portraying Ishikawa fishbone diagram and failure mode effect analysis (FMEA). The in vivo antidiabetic study was carried on induced diabetic rats. RESULTS: The optimised liquid SMEDDS-HES (OF) formulation showed emulsification time (Y 1) = 102.5 ± 2.52 s, globule size (Y 2) = 225.2 ± 3.40 nm, polydispersity index (Y 3) = 0.294 ± 0.62, and zeta potential (Y 4) = -25.4 ± 1.74 mV, respectively. The solid SMEDDS-HES (SOF-7) formulation was characterised by FTIR, PXRD, DSC, and SEM. The shelf life of SOF-7 was found to be 32.88 months. The heamatological and histopathological data of diabetic rats showed prominent antidiabetic activity. CONCLUSIONS: The optimised formulation showed improved dissolution, desired stability, and promising antidiabetic activity.

Mangiferin and Hesperidin Transdermal Distribution and Permeability through the Skin from Solutions and Honeybush Extracts (Cyclopia sp.)-A Comparison Ex Vivo Study.

Polyphenolic compounds-mangiferin and hesperidin-are, among others, the most important secondary metabolites of African shrub Cyclopia sp. (honeybush). The aim of this study was to compare the percutaneous absorption of mangiferin and hesperidin from solutions (water, ethanol 50%, (v/v)) and extracts obtained from green and fermented honeybush (water, ethanol 50%, (v/v)). Research was performed with the Bronaugh cells, on human dorsal skin. The mangiferin and hesperidin distributions in skin layers (stratum corneum, epidermis, and dermis) and in acceptor fluid (in every 2, 4, 6, and 24 h) were evaluated by HPLC-Photodiode Array Coulometric and Coulometric Electrochemical Array Detection. The transdermal distribution of hesperidin was also demonstrated by fluorescence microscopy. Results indicated that mangiferin and hesperidin were able to cross the stratum corneum and penetrate into the epidermis and dermis. An advantage of hesperidin penetration into the skin from the water over ethanol solution was observed (451.02 ± 14.50 vs. 357.39 ± 4.51 ng/cm2), as well as in the mangiferin study (127.56 ± 9.49 vs. 97.23 ± 2.92 ng/cm2). Furthermore, mangiferin penetration was more evident from nonfermented honeybush ethanol extract (189.85 ± 4.11 ng/cm2) than from solutions. The permeation of mangiferin and hesperidin through the skin to the acceptor fluid was observed regardless of whether the solution or the honeybush extract was applied. The highest ability to permeate the skin was demonstrated for the water solution of hesperidin (250.92 ± 16.01 ng/cm2), while the hesperidin occurring in the extracts permeated in a very low capacity. Mangiferin from nonfermented honeybush ethanol extract had the highest ability to permeate to the acceptor fluid within 24 h (152.36 ± 8.57 ng/cm2).

Hesperetin protects against palmitate-induced cellular toxicity via induction of GRP78 in hepatocytes.

Lipotoxicity plays a critical role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Hesperetin, a flavonoid derivative, has anti-oxidant, anti-inflammatory and cytoprotective properties. In the present study, we aim to examine whether hesperetin protects against palmitate-induced lipotoxic cell death and to investigate the underlying mechanisms in hepatocytes. Primary rat hepatocytes and HepG2 cells were pretreated with hesperetin for 30 min and then exposed to palmitate (1.0 mmol/L in primary rat hepatocytes; 0.5 mmol/L in HepG2 cells) in the presence or absence of hesperetin. Necrotic cell death was measured via Sytox green nuclei staining and quantified by LDH release assay. Apoptotic cell death was determined by caspase 3/7 activity and the protein level of cleaved-PARP. The unfolded protein response (UPR) was assessed by measuring the expression of GRP78, sXBP1, ATF4 and CHOP. Results show that hesperetin (50 µmol/L and 100 µmol/L) protected against palmitate-induced cell death and inhibited palmitate-induced endoplasmic reticulum (ER) stress in both primary rat hepatocytes and HepG2 cells. Hesperetin (100 µmol/L) significantly activated sXBP1/GRP78 signaling, whereas a high concentration of hesperetin (200 µmol/L) activated p-eIF2α and caused hepatic cell death. Importantly, GRP78 knockdown via siRNA abolished the protective effects of hesperetin in HepG2 cells. In conclusion, hesperetin protected against palmitate-induced hepatic cell death via activation of the sXBP1/GRP78 signaling pathway, thus inhibiting palmitate-induced ER stress. Moreover, high concentrations of hesperetin induce ER stress and subsequently cause cell death in hepatocytes.

Effects of hesperidin on the growth performance, antioxidant capacity, immune responses and disease resistance of red swamp crayfish (Procambarus clarkii).

We evaluated the effects of hesperidin on the nonspecific immunity, antioxidant capacity and growth performance of red swamp crayfish (Procambarus clarkii). A total of 900 healthy crayfish were randomly divided into six groups: the control group (fed the basal diet) and the HES25, HES50, HES75, HES100 and HES150 groups, which were fed the basal diet supplemented with 25, 50, 75, 100 and 150 mg kg-1 hesperidin, respectively. The feeding experiment lasted 8 weeks. The results indicated that compared with the control group, the crayfish groups supplemented with 50-150 mg kg-1 hesperidin had a decreased feed conversion ratio (FCR) and increased final body weight (FBW), specific growth rate (SGR) and weight gain (WG) (P < 0.05). The protein carbonyl content (PCC), reactive oxygen species (ROS) level and malondialdehyde (MDA) level in the hepatopancreas and hemocytes were significantly lower, while the total antioxidant capacity (T-AOC), glutathione peroxidase (GPx) activity, and superoxide dismutase (SOD) activity were significantly higher in the crayfish groups supplemented with 50-150 mg kg-1 hesperidin than in the control group. Supplementation with 50-150 mg kg-1 hesperidin significantly increased the activities of acid phosphatase (ACP), alkaline phosphatase (AKP), lysozyme (LZM), and phenoloxidase (PO) compared with the control group (P < 0.05); upregulated the mRNA expression of cyclophilin A (CypA), extracellular copper-zinc superoxide dismutase (ecCuZnSOD), GPxs, crustin, astacidin, Toll3 and heat shock protein 70 (HSP70) (P < 0.05); and decreased crayfish mortality following white spot syndrome virus (WSSV) infection. These findings indicate that dietary hesperidin supplementation at an optimum dose of 50-150 mg kg-1 may effectively improve nonspecific immunity, antioxidant capacity and growth performance in crayfish.

Hesperidin reduces adverse symptomatic intracerebral hemorrhage by promoting TGF-ß1 for treating ischemic stroke using tissue plasminogen activator.

Treatment with recombinant tissue plasminogen activator (rt-PA) is the most effective therapeutic option against brain ischemic stroke at the present time. However, elevated incidence of symptomatic intracerebral hemorrhage (SIH) greatly hinders ideal treatment outcome of rt-PA. We sought to assess the impacts of hesperidin on SIH following rt-PA therapies. Patients with ischemic stroke were assigned into two groups in a random fashion, to receive either rt-PA + placebo (Pc) or rt-PA + hesperidin. Treatment outcome was evaluated 24 h after the initial reperfusion using the transcranial Doppler ultrasonography (TCD) and the NIH Stroke Scale (NIHSS). Further, serum concentrations of transforming growth factor (TGF)-ß1, matrix metalloproteinase (MMP)-2, and MMP-9 were examined. Following the initial administration, stroke patients continued to receive either daily Pc or daily hesperidin, and the treatment outcome after 7 days was examined using the TCD, NIHSS, Glasgow Outcome Scale (GOS), and the Modified Rankin Scale (MRS). Combined treatment of rt-PA with hesperidin yielded significant improvement of outcomes, as revealed by better TCD and NIHSS scores as well as decreased SIH incidences, which could be attributable to elevation of TGF-ß1 and reduction in serum levels of both MMP-2 and MMP-9 caused by hesperidin. Follow-up hesperidin treatment for 7 consecutive days also markedly enhanced the recovery of stroke patients, as indicated by TCD, MRS, GOS, and NIHSS. Findings of the present study strongly suggested potential clinical application of hesperidin supplement in rt-PA therapies to reduce SIH and thereby improve the treatment outcomes of rt-PA in patients with ischemic stroke.

Hesperidin Induces ROS-Mediated Apoptosis along with Cell Cycle Arrest at G2/M Phase in Human Gall Bladder Carcinoma.

A natural predominant flavonoid hesperidin rich in citrus fruits exhibits multifunctional medicinal properties. The anticancerous potential of hesperidin has been widely explored; however, the gall bladder carcinoma (GBC) still remains untouched due to the unavailability of efficient experimental model. The aim of our study was to identify the apoptotic and antiproliferative potential of hesperidin in GBC. The promising efficacy of hesperidin was assessed through the generation of reactive oxygen species (ROS), cellular apoptosis, and loss of mitochondrial membrane potential (MMP) in the primary cells generated from surgically removed cancerous gall bladder tissues. Moreover, cell cycle analysis and caspases-3 activity were performed to confirm the apoptosis inducing potential of hesperidin. Results revealed that hesperidin exposure for 24 h at a dose of 200 µM reduced the cell proliferation of GBC cells significantly. In addition, hesperidin treatment further resulted in an increased ROS generation and nuclear condensation at the same dose. Caspase-3 activation and cell cycle arrest at G2/M phase were also accelerated in a dose-dependent manner. Together, these results suggest that hesperidin can be considered as a potential anticancerous compound for the treatment of GBC. Furthermore, evaluation of the pharmacological aspects of hesperidin is desirable for drug development.

Inhibition of Atherosclerotic Plaque Development by Oral Administration of α-Glucosyl Hesperidin and Water-Dispersible Hesperetin in Apolipoprotein E Knockout Mice.

OBJECTIVE: Hesperidin, an abundant flavonoid in citrus fruit, and its aglycone, hesperetin, have been reported to possess various physiological activities, including antioxidant, anti-inflammatory, hypolipidemic, and antihypertensive activities. In this study, we investigated whether α-glucosyl hesperidin and water-dispersible hesperetin have protective effects on atherosclerotic progression in apolipoprotein E knockout (Apo-E KO) mice. METHODS: Ten-week-old male Apo-E KO mice were randomly assigned a regular high-fat diet, a high-fat diet with 0.5% α-glucosyl hesperidin, or a high-fat diet with 0.1% water-dispersible hesperetin for 12 weeks. Measurement of plasma total cholesterol levels, histological staining of aortic root, and immunohistochemistry for macrophages were performed to evaluate atherosclerotic plaque formation. Vascular reactivity of mouse aortic rings was also measured. RESULTS: Both α-glucosyl hesperidin and water-dispersible hesperetin reduced plasma total cholesterol level. They also reduced plaque formation area, adipose deposition, and macrophage infiltration into atherosclerotic lesion. Vascular-endothelium-dependent relaxation in response to acetylcholine was improved in both experimental diet groups compared to the high-fat diet group. CONCLUSIONS: Our study suggests that both α-glucosyl hesperidin and water-dispersible hesperetin exert protective effects on atherosclerotic progression in Apo-E KO mice because they exhibit hypolipidemic activity, reduce inflammation through macrophages, and prevent endothelial dysfunction.

Hesperetin protects crayfish Procambarus clarkii against white spot syndrome virus infection.

Hesperetin is a natural flavanone compound, which mainly exists in lemons and oranges, and has potential antiviral and anticancer activities. In this study, hesperetin was used in a crayfish pathogen challenge to discover its effects on the innate immune system of invertebrates. The crayfish Procambarus clarkii was used as an experimental model and challenged with white spot syndrome virus (WSSV). Pathogen challenge experiments showed that hesperetin treatment significantly reduced the mortality caused by WSSV infection, while the VP28 copies of WSSV were also reduced. Quantitative reverse transcriptase polymerase chain reaction revealed that hesperetin increased the expression of several innate immune-related genes, including NF-kappaB and C-type lectin. Further analysis showed that hesperetin treatment plays a positive effects on three immune parameters like total hemocyte count, phenoloxidase and superoxide dismutase activity. Nevertheless, whether or not infected with WSSV, hesperetin treatment would significantly increase the hemocyte apoptosis rates in crayfish. These results indicated that hesperetin could regulate the innate immunity of crayfish, and delaying and reducing the mortality after WSSV challenge. Therefore, the present study provided novel insights into the potential therapeutic or preventive functions associated with hesperetin to regulate crayfish immunity and protect crayfish against WSSV infection, provide certain theoretical basis for production practice.

Dietary intake of the citrus flavonoid hesperidin affects stress-resilience and brain kynurenine levels in a subchronic and mild social defeat stress model in mice.

Depressive disorders are partly caused by chronic inflammation through the kynurenine (KYN) pathway. Preventive intervention using anti-inflammatory reagents may be beneficial for alleviating the risk of depression. In this study, we focused on the Japanese local citrus plant, Citrus tumida hort. ex Tanaka (C. tumida; CT), which contains flavonoids such as hesperidin that have anti-inflammatory actions. The dietary intake of 5% immature peels of CT fruits slightly increased stress resilience in a subchronic and mild social defeat (sCSDS) model in mice. Moreover, the dietary intake of 0.1% hesperidin significantly increased stress resilience and suppressed KYN levels in the hippocampus and prefrontal cortex in these mice. In addition, KYN levels in the hippocampus and prefrontal cortex were significantly correlated with the susceptibility to stress. In conclusion, these results suggest that dietary hesperidin increases stress resilience by suppressing the augmentation of KYN signaling under sCSDS.

Acute and sub-chronic oral toxicity studies of hesperidin isolated from orange peel extract in Sprague Dawley rats.

Citrus sinensis contains glycoside hesperetin-7-rhamnoglucoside (hesperidin) which harbor an array of therapeutic potentials including antioxidant, anticancer, and anti-inflammatory. However, a systematic examination of safety is needed before its utilization. Hence, the present investigation is aimed to evaluate acute and sub-chronic toxicity of hesperidin isolated from the citrus fruit. Hesperidin (73%) was isolated from a methanolic extract of dried peel of the citrus fruit, characterized using FTIR, and standardized by HPLC. Its acute oral toxicity (AOT) and sub-chronic toxicity studies were carried out in Sprague-Dawley rats. Hesperidin (5000 mg/kg) showed 10% mortality in AOT. In sub-chronic toxicity study, hesperidin (250 and 500 mg/kg) did not induce any abnormalities in body weight, food consumption, clinical signs, ophthalmological and neurological observations, urine analysis, hematology, clinical chemistry, organ weights, and gross pathology. However, hesperidin (1000 mg/kg) showed significant (p < 0.05) alterations in body and organ weights, hematology, clinical chemistry, and tissue histopathology. To conclude, hesperidin has median lethal dose (LD50) of 4837.5 mg/kg, and Low Observed Adverse Effect Level (LOAEL) at 1000 mg/kg for both male and female Sprague-Dawley rats. Thus, hesperidin isolated from citrus fruit showed a good safety profile in animal study.

Flavonoids protect colon against radiation induced colitis.

Radiation induced colitis is one the most common clinical issue for patients receiving radiotherapy. For this reason, we aimed to investigate the effect of antioxidant-effective flavonoids hesperidin and quercetin on the intestinal damage induced by radiation in this study. TNF-alpha, interleukin-10 (IL-10), heat shock protein 70 (HSP 70) and caspase 3, 8, 9 markers of apoptotic pathways were measured in the colon tissues of irradiated acute intestinal damage by enzyme-linked immunosorbent assay (ELISA). Irradiation of rats caused a significance increase of TNF-alpha, caspase 3/8/9 and decrease of IL-10 concentrations. Hesperidin and quercetin treatment resulted in decreased levels of TNF-alpha and increased levels of IL-10. Quercetin significantly decreased caspase 3/8/9 levels. Hesperidin produced a decreased of caspase 3/8/9 levels compared with irradiation group but this was statistically not significant. Only significant alteration of HSP 70 were seen in hesperidin treated rats. Further studies are needed to elucidate the mechanism by which flavonoids induced signaling provides protection against apoptosis and inflammation.

The effects of hesperidin or naringin dietary supplementation on the milk properties of dairy ewes.

BACKGROUND: Fortification of animal products with natural bioactive compounds appears to improve their quality and protects consumers against oxidation effects. An experiment was therefore conducted to examine the effects of hesperidin or naringin on milk oxidative stability, yield, composition, coagulation properties, and the fatty acid profile in dairy sheep. Thirty-six Chios ewes were assigned to four groups. A control group was fed a concentrate diet without supplementation. The other three groups were provided with the same diet further supplemented with hesperidin (6000 mg kg-1 ), naringin (6000 mg kg-1 ), or α-tocopheryl acetate (200 mg kg-1 ). The efficacy of flavonoids after a change in diet composition that lowered milk oxidation values by itself was also tested. The duration of the experiment was 28 days with alfalfa hay being the only forage source for the first 14 days, whereas after the 15th day a mixture of alfalfa hay and wheat straw (65:35) was provided. RESULTS: The oxidative stability of milk was improved after 14 days of addition of the examined flavonoids (P < 0.05). Milk malondialdehyde (MDA) values were also decreased as a result of flavonoid dietary supplementation, 14 days after the modification of the forage source. On the other hand, no significant differences in yield, chemical composition, coagulation properties, and fatty acid profile of ewe milk were observed among the treatments throughout the experiment. CONCLUSION: Enrichment of dairy ewes' diets with hesperidin and naringin might be effective in improving milk's oxidative stability without any effects on the milk's chemical composition, coagulation properties, and fatty acid profile. © 2019 Society of Chemical Industry.

Influence of citrus flavonoids on laying hen performance, inflammatory immune response, egg quality and yolk oxidative stability.

1. The objective of this study was to investigate the effects of dietary supplementation with natural flavonoids (naringin and hesperidin) on laying hens' performance, cellular immunity and egg quality parameters. 2. A total of 72 individually caged laying hens were allocated into 1 of 6 treatment groups: a control (C) group that was fed with a basal diet and groups that were offered the same diet further supplemented with hesperidin at 0.75 g/kg (E1), or 1.5 g/kg (E2), or naringin at 0.75 g/kg (N1), or 1.5 g/kg (N2) or α-tocopheryl acetate at 0.2 mg/kg (VE) for 67 d. 3. Supplementation with naringin or hesperidin did not affect the performance and egg quality (P > 0.05) apart from an improvement in the yolk colour that was more orange in naringin and hesperidin groups in comparison to the controls (P-linear < 0.05). Egg yolk and plasma cholesterol levels were not affected by citrus flavonoids (P-linear > 0.05). 4. Inflammatory immune response, measured by phytohaemagglutinin skin test (PHA), was suppressed in laying hens that were fed with either naringin (P-linear < 0.05) or hesperidin (P < 0.05). Egg yolk oxidative stability was improved from the 4th d after naringin or hesperidin supplementation. This beneficial effect was comparable to that of α-tocopheryl acetate and was observed in eggs that were stored for up to 120 d. 5. In conclusion, naringin and hesperidin may favourably prolong the shelf life of eggs, appear to possess anti-inflammatory properties and could improve the yolk colour without any side effects on the performance or egg quality traits.