Characterization of the antioxidant activity of aglycone and glycosylated derivatives of hesperetin: an in vitro and in vivo study

The flavonoids are mainly present in Citrus fruits as their glycosyl derivatives. This study was conducted comparing in vitro xanthine oxidase inhibitory activity of the aglycone hesperetin and its glycosylated forms (hesperidin and G‐hesperidin) and their effects on the plasma lipid profile and the oxidative–antioxidative system (TBARS and antioxidant enzymes) in rats. The concentrations of the major conjugated metabolites in rat plasma after oral administration of these compounds were also determined. Wistar male rats were randomly assigned to three groups (n = 6) supplemented for 30 days with 1 mmol/kg body mass of hesperetin, hesperidin or G‐hesperidin. Hesperetin was a stronger xanthine oxidase inhibitor (IC₅₀ = 53 μM and K_i = 17.3 μM) than the glycosylate derivatives. Supplementation with the three compounds led to a lower (more favorable) atherogenic index, and an antioxidant preventive effect from the increase of hepatic superoxide dismutase was observed associated to HT supplementation, possibly because of the higher level of hesperetin‐glucuronide in rat plasma. Copyright © 2015 John Wiley & Sons, Ltd.     

Hesperidin and its aglycone hesperetin in breast cancer therapy: A review of recent developments and future prospects

Breast cancer (BC) has high incidence and mortality rates, making it a major global health issue. BC treatment has been challenging due to the presence of drug resistance and the limited availability of therapeutic options for triple-negative and metastatic BC, thereby urging the exploration of more effective anti-cancer agents. Hesperidin and its aglycone hesperetin, two flavonoids from citrus species, have been extensively evaluated for their anti-cancer potentials. In this review, available literatures on the chemotherapeutic and chemosensitising activities of hesperidin and hesperetin in preclinical BC models are reported. The safety and bioavailability of hesperidin and hesperetin as well as the strategies to enhance their bioavailability are also discussed. Overall, hesperidin and hesperetin can inhibit cell proliferation, migration and BC stem cells as well as induce apoptosis and cell cycle arrest in vitro. They can also inhibit tumour growth, metastasis and neoplastic changes in tissue architecture in vivo. Moreover, the co-administration of hesperidin or hesperetin with doxorubicin, letrozole or tamoxifen can enhance the efficacies of these clinically available agents. These chemotherapeutic and chemosensitising activities of hesperidin and hesperetin have been linked to several mechanisms, including the modulation of signalling pathways, glucose uptake, enzymes, miRNA expression, oxidative status, cell cycle regulatory proteins, tumour suppressor p53, plasma and liver lipid profiles as well as DNA repair mechanisms. However, poor water solubility, extensive phase II metabolism and apical efflux have posed limitations to the bioavailability of hesperidin and hesperetin. Various strategies for bioavailability enhancement have been studied, including the utilisation of nano-based drug delivery systems and the co-administration of hesperetin with other flavonoids. In particular, nanoformulated hesperidin and hesperetin possess greater chemotherapeutic and chemosensitising activities than free compounds. Despite promising preclinical results, further safety and efficacy evaluation of hesperidin and hesperetin as well as their nanoformulations in clinical trials is required to ascertain their potentials to be developed as clinically useful agents for BC treatment.     

Hesperetin glucuronides induce adipocyte differentiation via activation and expression of peroxisome proliferator-activated receptor-γ

In previous reports, hesperidin, a flavonoid glucoside from citrus fruit, is hydrolyzed to hesperetin, an aglycone of hesperidin, and converted to the hesperetin glucuronides (H7-OG and H3′-OG) in vivo and depresses blood glucose levels. But there are no reports on the activity of hesperetin glucuronides. To determine the activity of hesperetin glucuronides, H7-OG and H3′-OG were synthesized and peroxisome proliferator-activated receptor-γ (PPARγ) agonist activity was observed at 250?μM. These glucuronides accelerated the differentiation of 3T3-L1 cells into adipocytes at 10?μM. Furthermore, H7-OG showed additive effects in reporter gene assays and caused noncompetitive reactions in time-resolved fluorescence resonance energy transfer assays with a thiazolidinedione derivative. Our results indicated that hesperetin glucuronides activated PPARγ, accelerated adipocyte differentiation.     

Transepithelial transport of hesperetin and hesperidin in intestinal Caco-2 cell monolayers

The cell permeability of hesperetin and hesperidin, anti-allergic compounds from citrus fruits, was measured using Caco-2 monolayers. In the presence of a proton gradient, hesperetin permeated cells in the apical-to-basolateral direction at the rate (Jap-->bl) of 10.43+/-0.78 nmol/min/mg protein, which was more than 400-fold higher than that of hesperidin (0.023+/-0.008 nmol/min/mg protein). The transepithelial flux of hesperidin, both in the presence or absence of a proton gradient, was nearly the same and was inversely correlated with the transepithelial electrical resistance (TER), indicating that the transport of hesperidin was mainly via paracellular diffusion. In contrast, the transepithelial flux of hesperetin was almost constant irrespective of the TER. Apically loaded NaN3 or carbonyl cyanide m-chlorophenylhydrazone (CCCP) decreased the Jap-->bl of hesperetin, in the presence of proton gradient, by one-half. In the absence of a proton gradient, both Jap-->bl and Jbl-->ap of hesperetin were almost the same (5.75+/-0.40 and 5.16+/-0.73 nmol/min/mg protein). Jbl-->ap of hesperetin in the presence of a proton gradient was lower than Jbl-->ap in the absence of a proton gradient. Furthermore, Jbl-->ap in the presence of a proton gradient remarkably increased upon addition of NaN3 specifically to the apical side. These results indicate that hesperetin is absorbed by transcellular transport, which occurs mainly via proton-coupled active transport, and passive diffusion. Thus, hesperetin is efficiently absorbed from the intestine, whereas hesperidin is poorly transported via the paracellular pathway and its transport is highly dependent on conversion to hesperetin via the hydrolytic action of microflora. We have given novel insight to the absorption characteristics of hesperetin, that is proton-coupled and energy-dependent polarized transport.     

Bioavailability of glucosyl hesperidin in rats

Glucosyl hesperidin (G-hesperidin) is a water-soluble derivative of hesperidin. We compared the absorption and metabolism of G-hesperidin with those of hesperidin in rats. After oral administration of G-hesperidin or hesperidin to rats, hesperetin was detected in sera hydrolyzed with beta-glucuronidase, but it was not detectable in unhydrolyzed sera. Serum hesperetin was found more rapidly in rats administered G-hesperidin than in those administered hesperidin. The area under the concentration-time curve for hesperetin in the sera of rats administered G-hesperidin was approximately 3.7-fold greater than that of rats administered hesperidin. In the urine of both administration groups, hesperetin and its glucuronide were found. Urinary excretion of metabolites was higher in rats administered G-hesperidin than in those administered hesperidin. These results indicate that G-hesperidin presents the same metabolic profile as hesperidin. Moreover, it was concluded that G-hesperidin is absorbed more rapidly and efficiently than hesperidin, because of its high water solubility.     

Bioavailability of Glucosyl Hesperidin in Rats

Glucosyl hesperidin (G-hesperidin) is a water-soluble derivative of hesperidin. We compared the absorption and metabolism of G-hesperidin with those of hesperidin in rats. After oral administration of G-hesperidin or hesperidin to rats, hesperetin was detected in sera hydrolyzed with β-glucuronidase, but it was not detectable in unhydrolyzed sera. Serum hesperetin was found more rapidly in rats administered G-hesperidin than in those administered hesperidin. The area under the concentration-time curve for hesperetin in the sera of rats administered G-hesperidin was approximately 3.7-fold greater than that of rats administered hesperidin. In the urine of both administration groups, hesperetin and its glucuronide were found. Urinary excretion of metabolites was higher in rats administered G-hesperidin than in those administered hesperidin. These results indicate that G-hesperidin presents the same metabolic profile as hesperidin. Moreover, it was concluded that G-hesperidin is absorbed more rapidly and efficiently than hesperidin, because of its high water solubility.     

Hesperetin-etoposide combinations induce cytotoxicity in U2OS cells: Implications on therapeutic developments for osteosarcoma

Osteosarcoma chemotherapy has improved survival rates, however, chemoresistance and drug toxicity still limit therapy. Drug combinations may overcome these limitations by allowing fewer chemoresistant cells to survive. The aim of this study was to evaluate the cytotoxic potential of hesperetin to osteosarcoma and to analyze the cell cycle effects of combinations of hesperetin with chemotherapeutic agents. For this, the U2OS human osteosarcoma cell line was exposed to hesperetin or hesperetin combined with etoposide or doxorubicin in defined proportions. Hesperetin was less cytotoxic compared to chemotherapeutic agents, as shown by cell growth, viability and clonogenic assays. Notwithstanding, hesperetin combined with etoposide showed additive effects on the inhibition of cell growth. Furthermore, hesperetin induced G2-phase arrest, associated with decreased gene expression of cyclins B1 and E1 and cyclin-dependent kinases 1 and 2. The combination with higher additive effect resulted in higher percentage of cells in G2-phase, showing that G2-phase arrest is associated with cytotoxicity. Moreover, hesperetin induced cytostatic effects. In conclusion, our results suggest that G2-phase arrest is an important step for hesperetin-induced cytotoxicity in U2OS cells. Hesperetin shows potential cytotoxicity when combined with etoposide, which may have implications on therapeutic developments for osteosarcoma.     

High-performance liquid chromatography methods for the analysis of adrenergic amines and flavanones in Citrus aurantium L. var. amara

Reverse-phase HPLC coupled with photodiode array detection was used for the simultaneous separation and determination of naturally occurring adrenergic amines (octopamine, synephrine and tyramine) in fruits and dry extracts of Citrus aurantium L. var. amara and in herbal medicines derived therefrom. Synephrine was the main component in fruits (0.10-0.35%) and in dry extracts (3.00-3.08%) and was present in the range 0.25-0.99% in herbal medicines. Flavanones were analysed in the same samples using a reverse-phase HPLC technique which allowed the identification and quantification of neoeriocitrin, narirutin, naringin, hesperidin, neohesperidin, naringenin and hesperetin. C. aurantium fruits and derivatives contained mainly glycosylated flavanones: in particular, naringin and neohesperidin were found to be the major flavonoids and their concentrations ranged from 1.80 to 26.30 and from 3.90 to 14.71 mg/g, respectively. The levels of aglycones were very low in all samples tested.     

枳实提取物及其药效组分对OX—LDL损伤的人脐静脉内皮细胞ICAM-1表达和NO释放的影响

目的：研究枳实提取物及其药效组分橙皮苷和新橙皮苷对氧化低密度脂蛋白（oxidized lowd ensity lipoprotein,Ox—LDL）损伤的人脐静脉内皮细胞（human umbilical vein endothelial cells line,HUVEC）细胞间黏附分子-1（intercellular adhesion molecule-1,ICAM-1）表达和一氧化氮（nitric oxide,NO）释放的影响。方法：体外培养HUVEC,50μg／mLOX—LDL制造HUVEC损伤模型。以MTS染色法检测细胞毒性确定用药浓度。细胞ELISA法测定细胞表面ICAM-1的含量,试剂盒测定细胞培养上清液中NO含量。结果：①枳实提取物小于等于2mg／mL时,橙皮苷浓度小于等于0．03125mg／mL时,新橙皮苷浓度小于等于0．25mg／mL时,HUVEC存活率分别大于80％。②2．0mg／mL和1．0mg／mL两个浓度的枳实提取物、15．625μg／mL的橙皮苷和0．2500mg／mL新橙皮苷对OX—LDL诱导的HUVEC的ICAM-1表达有显著抑制作用。③2．0mg／mL枳实提取物显著提高OX—LDL诱导的HUVEC和正常HUVEC培养液中的NO含量;7．813ixg／mL、15．625μg／mL和31．250μg／mL 3个浓度的橙皮苷能显著提高OX—LDL诱导的HUVEC培养液中的NO含量,31．250μg／mL的橙皮苷能促进正常HUVEC的NO释放;0．2500mg／mL和0．1250mg／mL 2个浓度的新橙皮苷能显著提高OX—LDL诱导的HUVEC培养液中的NO含量。结论：枳实提取物及其药效组分橙皮苷、新橙皮苷能抑制Ox-LDL诱导的HUVEC的ICAM-1表达,促进Ox-LDL诱导的HUVEC的NO释放。     

Molecular mechanisms of luteolin-7-O-glucoside-induced growth inhibition on human liver cancer cells: G2/M cell cycle arrest and caspase-independent apoptotic signaling pathways

Luteolin-7-O-glucoside (LUT7G), a flavone subclass of flavonoids, has been found to increase anti-oxidant and anti-inflammatory activity, as well as cytotoxic effects. However, the mechanism of how LUT7G induces apoptosis and regulates cell cycles remains poorly understood. In this study, we examined the effects of LUT7G on the growth inhibition of tumors, cell cycle arrest, induction of ROS generation, and the involved signaling pathway in human hepatocarcinoma HepG2 cells. The proliferation of HepG2 cells was decreased by LUT7G in a dose-dependent manner. The growth inhibition was due primarily to the G2/M phase arrest and ROS generation. Moreover, the phosphorylation of JNK was increased by LUT7G. These results suggest that the anti-proliferative effect of LUT7G on HepG2 is associated with G2/M phase cell cycle arrest by JNK activation. [BMB Reports 2013; 46(12): 611-616]     

Transepithelial transport of hesperetin and hesperidin in intestinal Caco-2 cell monolayers

The cell permeability of hesperetin and hesperidin, anti-allergic compounds from citrus fruits, was measured using Caco-2 monolayers. In the presence of a proton gradient, hesperetin permeated cells in the apical-to-basolateral direction at the rate (J_ap → bl) of 10.43 ± 0.78 nmol/min/mg protein, which was more than 400-fold higher than that of hesperidin (0.023 ± 0.008 nmol/min/mg protein). The transepithelial flux of hesperidin, both in the presence or absence of a proton gradient, was nearly the same and was inversely correlated with the transepithelial electrical resistance (TER), indicating that the transport of hesperidin was mainly via paracellular diffusion. In contrast, the transepithelial flux of hesperetin was almost constant irrespective of the TER. Apically loaded NaN₃ or carbonyl cyanide m-chlorophenylhydrazone (CCCP) decreased the J_ap → bl of hesperetin, in the presence of proton gradient, by one-half. In the absence of a proton gradient, both J_ap → bl and J_bl → ap of hesperetin were almost the same (5.75 ± 0.40 and 5.16 ± 0.73 nmol/min/mg protein). J_bl → ap of hesperetin in the presence of a proton gradient was lower than J_bl → ap in the absence of a proton gradient. Furthermore, J_bl → ap in the presence of a proton gradient remarkably increased upon addition of NaN₃ specifically to the apical side. These results indicate that hesperetin is absorbed by transcellular transport, which occurs mainly via proton-coupled active transport, and passive diffusion. Thus, hesperetin is efficiently absorbed from the intestine, whereas hesperidin is poorly transported via the paracellular pathway and its transport is highly dependent on conversion to hesperetin via the hydrolytic action of microflora. We have given novel insight to the absorption characteristics of hesperetin, that is proton-coupled and energy-dependent polarized transport.     

Flavonoids in Citrus Hybrids

The occurrence and distribution of flavanone glycosides in the leaves and fruits of many kinds of artificial citrus hybrid plants were investigated by polyamide thin-layer chromatography. The citrus hybrids can be divided into two broad categories, a) those containing rutinosyl glycosides, b) those containing neohesperidosyl glycosides in accordance with the case of natural citrus species. The fiavonoid patterns of rutinosyl glycosides are classified into the following groups, a) hesperidin, b) narirutin, c) hesperidin and narirutin, d) didymin and narirutin, e) hesperidin, narirutin and eriocitrin and f) hesperidin and eriocitrin, while the pattern of neohesperidosyl glycosides fall into six groups, a) naringin, b) neohesperidin and naringin, c) neohesperidin, naringin and neoeriocitrin, d) neohesperidin and neoeriocitrin, e) naringin and neoeriocitrin, and f) poncirin, neohesperidin, naringin and neoeriocitrin. It is worthy of note that a hybrid (accession number 1088) between C. unshiu and C. hassaku contains only narirutin. Among the ninty-four hybrids examined, fifty-three varieties were obviously different from female parents in their flavonoid pattern and could be judged as true hybrids by fiavonoids but the others could not.

Additionally, a survey of fiavonoids in newly found natural pummelo- and Daidai hybrids were carried out in connection with their origin.     

Anti-aging effects of hesperidin on Saccharomyces cerevisiae via inhibition of reactive oxygen species and UTH1 gene expression

This study used a replicative lifespan assay of K6001 yeast to screen anti-aging food factors in commercial flavonoids. Hesperidin derived from the Citrus genus extended the lifespan of yeast at doses of 5 and 10 μM as compared with the control group (p<0.01, p<0.01). Reactive oxygen species (ROS), real-time PCR (RT-PCR), and lifespan assays of uth1 and skn7 mutants with the K6001 background were used to study the anti-aging mechanisms in yeast. The results indicate that hesperidin significantly inhibits the ROS of yeast, and UTH1 gene expression, and that SKN7 gene are involved in hesperidin-mediated lifespan extension. Further, increases in the Sir2 homolog, SIRT1 activity, and SOD gene expression were confirmed at doses of 5 (p<0.01) and 10 μM (p<0.05). This suggests that Sir2, UTH1 genes, and ROS inhibition after administration of hesperidin have important roles in the anti-aging effects of yeast. However, the aglycon hesperetin did not exhibit anti-aging effects in yeast.     

New potent antioxidative hydroxyflavanones produced with Aspergillus saitoi from flavanone glycoside in citrus fruit

Potent antioxidative hydroxyflavanones were produced with Aspergillus saitoi from hesperidin or naringin, which are flavanone glycosides in citrus fruit with weak antioxidative activity. The hydroxyflavanone produced from hesperidin was identified as 8-hydroxyhesperetin (8-HHE), a novel substance, and those from naringin were identified as carthamidin (6-hydroxynaringenin) and isocarthamidin (8-hydroxynaringenin) by FAB-MS, 1H-NMR and 13C-NMR analyses. The antioxidative activity of these hydroxyflavanones was examined by using the free radical-scavenging system of 1,1-diphenyl-2-picrylhydrazyl (DPPH) and the methyl linoleate oxidation system. The hydroxyflavanones (8-HHE, carthamidin, and isocarthamidin) exhibited stronger activity than the flavanone glycosides (hesperidin or naringin) and their aglycones (hesperetin or naringenin). The activity of 8-HHE and isocarthamidin was comparable to that of alpha-tocopherol, and that of carthamidin was weaker than that of isocarthamidin. The hydroxyflavanones, which were hydroxylated on A ring of flavanone by Aspergillus saitoi, were obtained as potent antioxidants.     

Effects of hesperetin on the production of inflammatory mediators in IL-1β treated human synovial cells

This study was conducted to evaluate the efficacy of hesperetin in regulating interleukin-1β (IL-1β)-induced production of the matrix metalloproteinase (MMP)-3 and IL-6 in human synovial cell line, SW982. Treatment with hesperetin at 1 or 10 μM significantly (P < 0.05) inhibited IL-1β-induced MMP-3 and IL-6 production when measured by enzyme-linked immunosorbent assay (ELISA). The effects of hesperetin on the activation of mitogen-activated protein kinases (MAPKs) were also examined in SW982 cells by ELISA assay. IL-1β-induced JNK activation was inhibited by hesperetin. These results suggest that hesperetin reduces the production of MMP and IL-6 in SW982 synovial cells by inhibiting JNK.     

Gomisin A ameliorates metastatic melanoma by inhibiting AMPK and ERK/JNK-mediated cell survival and metastatic phenotypes

BackgroundGomisin A (G.A), a lignan compound extracted from the fruits of Schisandra chinensis, is known to exert anti-tumor effects on hepatocarcinoma and colorectal cancer cells. Suppression of proliferation and metastatic abilities of cancer cells are some effective cancer treatment methods.PurposeThe objective of this study is to investigate the effects of G.A on metastatic melanoma, and the mechanism by which it affects metastatic melanoma.Study designThe anti-proliferative and anti-metastatic effects of G.A were observed in in vitro and in vivo.MethodsWST assay and flow cytometry were conducted to investigate the effect of G.A on proliferation, cell cycle arrest, and apoptosis in metastatic melanoma cell lines. Migration and invasion abilities of G.A-treated melanoma cells were observed by wound healing and invasion assays.ResultsG.A (25–100 μM) decreased the viability of melanoma cells by inducing cell cycle arrest and apoptosis. These anti-proliferative effects of G.A were found to be mediated by AMPK, ERK, and JNK activation. G.A (5–20 μM) decreased the migration and invasion of melanoma cells by suppressing epithelial-mesenchymal transition (EMT). Consequently, G.A (2–50 mg/kg) inhibited lung metastasis by suppressing EMT and inducing cell cycle arrest and apoptosis in melanoma cells.ConclusionThese results conclude that G.A has the potential to reduce metastatic melanoma through its anti-proliferative and anti-metastatic effects.     

Molecular mechanism of adaphostin-mediated G1 arrest in prostate cancer (PC-3) cells: signaling events mediated by hepatocyte growth factor receptor, c-Met, and p38 MAPK pathways

Adaphostin (NSC680410), a small molecule congener of tyrphostin AG957, has been demonstrated previously to have significant anti-proliferative effects in several leukemia models. However, this effect of adaphostin in adherent cells/solid tumor models has not been examined. In this study, we investigated the anti-proliferative effects of adaphostin in the human prostate cancer cell line PC-3. Specifically, we explored the potential molecular mechanism(s) by which adaphostin elicits its anti-proliferative effect(s). We demonstrate that adaphostin inhibits the proliferation of PC-3 cells by inducing a G(1) phase cell cycle arrest. This adaphostin-induced G(1) arrest was associated with an increase in the expression of p21 and p27 and a decrease in the expression of G(1)-specific cyclins (cyclin A, D1, and D3) and cyclin-dependent kinases 4 and 6. Consequently, a dramatic decrease in the phosphorylation of retinoblastoma protein was also observed. Additionally, we found that adaphostin treatment induced a decrease in the phosphorylation of nucleophosmin, a major nuclear phosphoprotein, and that this decreased phosphorylation was a result of the p21- and p27-mediated inactivation of cyclin E-cyclin-dependent kinase 2 complex kinase activity. Furthermore, we have determined that the adaphostin-mediated cell cycle arrest of PC-3 cells is dependent upon activation of the p38 MAPK. We also demonstrate that the hepatocyte growth factor receptor-c-Met is involved in the adaphostin-mediated signaling events that regulate p38 MAPK. Taken together, these results identify for the first time a signaling cascade of adaphostin-mediated G(1) phase-specific cell cycle arrest in PC-3 cells. These findings suggest that the tyrphostin member has a broader spectrum of activity than originally predicted.     

Tissue distribution of hesperetin in rats after a dietary intake

Hesperetin, the aglycone of hesperidin present in citrus fruits, possesses various biological activities. We assessed the tissue distribution of hesperetin in rats fed with a 0.2% hesperetin diet for 4 weeks. Its highest concentration was found in the liver, and the second highest was in the aorta. The aorta is assumed to be one of the main target tissues of hesperetin for exerting its functions.     

Tissue Distribution of Hesperetin in Rats after a Dietary Intake

Hesperetin, the aglycone of hesperidin present in citrus fruits, possesses various biological activities. We assessed the tissue distribution of hesperetin in rats fed with a 0.2% hesperetin diet for 4 weeks. Its highest concentration was found in the liver, and the second highest was in the aorta. The aorta is assumed to be one of the main target tissues of hesperetin for exerting its functions.