Instability of, and generation of hydrogen peroxide by, phenolic compounds in cell culture media

Many papers in the literature have described complex effects of flavonoids and other polyphenols on cells in culture. In this paper we show that hydroxytyrosol, delphinidin chloride and rosmarinic acid are unstable in three commonly-used cell culture media (Dulbecco’s modified Eagle’s medium (DMEM), RPMI 1640 (RPMI) and Minimal Essential Medium Eagle (MEM)) and undergo rapid oxidation to generate H₂O₂. This may have confounded some previous studies on the cellular effects of these compounds. By contrast, apigenin, curcumin, hesperetin, naringenin, resveratrol and tyrosol did not generate significant H₂O₂ levels in these media. Nevertheless, curcumin and, to a lesser extent, resveratrol (but not tyrosol) were also unstable in DMEM, so the absence of detectable H₂O₂ production by a compound in cell culture media should not be equated to stability of that compound. Compound instability and generation of H₂O₂ must be taken into account in interpreting effects of phenolic compounds on cells in culture.     

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.     

Glycosylation of hesperetin by plant cell cultures

The biotransformation of hesperetin by cultured cells of Ipomoea batatas and Eucalyptus perriniana was investigated. Three glycosides, hesperetin 3'-O-beta-D-glucopyranoside (33 microg/g fr. wt of cells), hesperetin 3',7-O-beta-D-diglucopyranoside (217 microg/g fr. wt of cells), and hesperetin 7-O-[6-O-(beta-D-glucopyranosyl)]-beta-d-glucopyranoside (beta-gentiobioside, 22 microg/g fr. wt of cells), together with three hitherto known glycosides, hesperetin 5-O-beta-d-glucopyranoside (23 microg/g fr. wt of cells), hesperetin 7-O-beta-D-glucopyranoside (57 microg/g fr. wt of cells), and hesperetin 7-O-[6-O-(alpha-L-rhamnopyranosyl)]-beta-D-glucopyranoside (beta-rutinoside, hesperidin, 13 microg/g fr. wt of cells), were isolated from cultured suspension cells of E. perriniana that had been treated with hesperetin. Oligosaccharide chains were regioselectively formed at the C-7 position of hesperetin to afford beta-gentiobioside and beta-rutinoside. On the other hand, cultured I. batatas cells converted hesperetin into hesperetin 3'-O-beta-D-glucopyranoside (60 microg/g fr. wt of cells), hesperetin 5-O-beta-D-glucopyranoside (23 microg/g fr. wt of cells), and hesperetin 7-O-beta-D-glucopyranoside (110 microg/g fr. wt of cells).     

Hesperetin Inhibit Adipocyte Differentiation and Enhance Bax‐ and p21‐Mediated Adipolysis in Human Mesenchymal Stem Cell Adipogenesis

We aimed to explore the antiadipogenic and adipolysis effect of hesperetin in human mesenchymal stem cells (hMSCs)–induced adipogenesis. IC₅₀ value of hesperetin was higher for hMSCs such as 149.2 ± 13.2 μmol for 24 h and 89.4 ± 11.4 μmol in 48 h, whereas in preadipocytes was 87.6 ± 9.5 μmol and 72.4 ± 5.6 μmol in 24 h and 48 h, respectively. Hesperetin treatment (5, 10, and 20 μmol) to adipogenesis‐induced hMSCs (Group 1) and preadipocytes (Group 2) resulted in a significantly (p < 0.05) increased lipolysis. The treatment with hesperetin decreased the expression of resistin, adiponectin, aP₂, LPL, PPAR‐γ, and TNF‐α in Groups 1 and 2, whereas a significant increase was observed in Bcl, Bax, and p21 expression in Group 2 compared to untreated preadipocytes. hMSCs cultured in adipogenic medium along with hesperetin significantly inhibited adipocyte differentiation and increased the proapoptotic gene expression levels in preadipocyte. Our result indicates the antiadipogenic and adipolysis effects of hesperetin.     

Modulatory efficacy of hesperetin (citrus flavanone) on xenobiotic-metabolizing enzymes during 1,2-dimethylhydrazine-induced colon carcinogenesis

Colorectal cancer is the second leading cause of cancer death worldwide with diet playing a prominent role in disease initiation and progression. Diet and nutrition play an important role during this multistep colon carcinogenic process. We have investigated the modulatory efficacy of hesperetin on aberrant crypt foci (ACF) and xenobiotic-metabolizing enzymes on 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis. Male albino Wistar rats were randomly divided into six groups. Group 1 served as control, received modified pellet diet and group 2 rats received 20 mg/kg body weight of hesperetin p.o. every day. Groups 3-6 rats were given subcutaneous injections of 1,2-dimethylhydrazine (20 mg/kg body weight) once a week for 15 weeks to induce ACF in the colon. In addition, rats in group 4 received hesperetin as in group 2 orally for the first 15 weeks (initiation), group 5 rats received hesperetin as in group 2 after the last injection of DMH and continued till the end of the experimental period (post-initiation). Group 6 received hesperetin as in group 2 throughout the entire period of 32 weeks. DMH exposure showed high incidence (90%) of ACF (280 ± 24.5 aberrant crypt/colon) and dysplastic ACF, elevated activities of phase I enzymes and reduced the activities of phase II enzymes in the liver and colonic mucosa of colon cancer bearing rats. Hesperetin supplementation significantly reversed these effects, the effect being more pronounced in group 6 rats (hesperetin supplemented throughout the study period).These findings suggest that hesperetin can significantly reduce the formation of preneoplastic lesions and effectively modulate the xenobiotic-metabolizing enzymes in rats.     

Inhibition of the formation of the neurotoxin 5-S-cysteinyl-dopamine by polyphenols

The death of nigral neurons in Parkinson's disease is thought to involve the formation of the endogenous neurotoxin, 5-S-cysteinyl-dopamine. In the present study, we show that the polyphenols, (+)-catechin and caffeic acid, which contain a catechol moiety, inhibit tyrosinase-induced formation of 5-S-cysteinyl-dopamine via their capacity to undergo tyrosinase-induced oxidation to yield cysteinyl-polyphenol adducts. In contrast, the inhibition afforded by the flavanone, hesperetin, was not accompanied by the formation of cysteinyl-hesperetin adducts, indicating that it may inhibit via direct interaction with tyrosinase. Whilst the stilbene resveratrol also inhibited 5-S-cysteinyl-dopamine formation, this was accompanied by the formation of dihydrobenzothiazine, a strong neurotoxin. Our data indicate that the inhibitory effects of polyphenols against 5-S-cysteinyl-dopamine formation are structure-dependent and shed further light on the mechanisms by which polyphenols exert protection against neuronal injury relevant to neurodegenerative diseases.     

Antioxidative effects of hesperetin against 7,12-dimethylbenz(a)anthracene-induced oxidative stress in mice

We investigated the effects of the chronic administration of hesperetin on the activation of the antioxidant defence system in mice in which oxidative stress had been induced by 7,12-dimethylbenz(a)anthracene (DMBA). Mice were divided randomly into three treatment groups. Hesperetin was administered orally to two of the three groups at 10 and 50 mg/kg body weight for 5 weeks. Subsequently, each group was subdivided randomly into DMBA-treated and untreated groups. The DMBA-treated groups were intragastrically administered a dose of 34 mg/kg BW in corn oil vehicle twice a week for 2 weeks. The TBARS value showed a tendency to decrease following hesperetin treatment; these decreases were significantly greater in the DMBA-treated than the untreated groups. Hesperetin significantly decreased the carbonyl content at the high dose in both DMBA-treated and untreated mice. Catalase and SOD activity were increased by hesperetin; this increase was more pronounced in DMBA-treated than untreated mice. Catalase, Mn-SOD, and CuZn-SOD expression analyses supported these results. Although the GSH-px and GR activity were little affected, hesperetin treatment significantly increased the GSH/GSSG ratio in the DMBA-treated group in a dose-dependent manner. These results suggest that hesperetin shows antioxidant activity and plays a protective role against DMBA-induced oxidative stress.     

Transepithelial transport of flavanone in intestinal Caco-2 cell monolayers

Our recent study [S. Kobayashi, S. Tanabe, M. Sugiyama, Y. Konishi, Transepithelial transport of hesperetin and hesperidin in intestinal Caco-2 cell monolayers, Biochim. Biophys. Acta, 1778 (2008) 33-41] shows that the mechanism of absorption of hesperetin involves both proton-coupled active transport and transcellular passive diffusion. Here, as well as analyzing the cell permeability of hesperetin, we also study the transport of other flavanones, naringenin and eriodictyol, using Caco-2 cell monolayers. Similar to hesperetin mentioned, naringenin and eriodictyol showed proton-coupled polarized transport in apical-to-basolateral direction in non-saturable manner, constant permeation in the apical-to-basolateral direction (J_ap → bl) irrespective of the transepithelial electrical resistance (TER), and preferable distribution into the basolateral side after apical loading in the presence of a proton gradient. Furthermore, the proton-coupled J_ap → bl of hesperetin, naringenin and eriodictyol, were inhibited by substrates of the monocarboxylic acid transporter (MCT), such as benzoic acid, but not by ferulic acid. In contrast, both benzoic and ferulic acids have no stimulatory effect on J_ap → bl of each flavanone by trans-stimulation analysis. These results indicates that proton-driven active transport is commonly participated in the absorption of flavanone in general, and that its transport is presumed to be unique other than MCT-mediated transport for absorption of phenolic acids (PAs), sodium-dependent MCT (SMCT) nor anion exchanger-mediated transport.     

Modulation of peroxynitrite-induced fibroblast injury by hesperetin: a role for intracellular scavenging and modulation of ERK signalling

Peroxynitrite is thought to contribute to the progression of many diseases including cardiovascular disease, cancer, and neurodegenerative disorders. We report that pre-treatment of fibroblasts with the citrus flavanone, hesperetin, prior to peroxynitrite exposure protects against peroxynitrite-mediated cytotoxicity. This protection was partially mediated by the intracellular scavenging of peroxynitrite by hesperetin as exposure of fibroblasts to peroxynitrite following hesperetin loading led to the formation of two intracellular nitro-hesperetin derivatives. In addition, protection appeared to be mediated by hesperetin-induced changes in MAP kinase signalling. Exposure of fibroblasts to hesperetin led to concentration-dependent increases in the phosphorylation of ERK1/2 and was observed to restore peroxynitrite-mediated decreases in ERK1/2 phosphorylation. We propose that the protective potential of hesperetin in fibroblasts may be mediated both by intracellular scavenging of peroxynitrite and by modulation of fibroblast signalling.     

DNA binding affinity and antioxidative activity of copper(II) and zinc(II) complexes with a novel hesperetin Schiff base ligand

A hesperetin Schiff base ligand (H₄L) and its complexes, [H₃CuL·OAc]·H₂O and [H₃ZnL·OAc]·2H₂O, have been synthesized and characterized on the basis of elemental analysis, molar conductivity, ¹H NMR, mass spectra, UV-Vis spectra and IR spectra. The binding of these two complexes and the ligand to DNA has been investigated by ultraviolet absorption spectroscopy, fluorescence spectroscopy and viscosity measurements. The experiments indicate that all the compounds can bind to DNA through an intercalative mode and the complexes intercalate into DNA more deeply than that of the ligand. In addition, the antioxidative activity was also determined. The 50% inhibition obtained for the ligand and its complexes demonstrates that, compared to the ligand, the complexes exhibit higher antioxidative activity in the suppression of and HO.