Hesperetin treatment attenuates glycation of lens proteins and advanced­glycation end products generation.

Advanced glycation end products (AGEs) in lens proteins increase with aging, thus inducing cataracts and/or presbyopia. Hesperetin (Hst), which is an abundant plant flavanone largely derived from citrus species, and its derivatives attenuate cataracts and presbyopia in vivo and in vitro; however, no reports have described its effects on AGE formation in lens proteins. The present study demonstrated that AGEs in lens proteins increase with age in mice. Additionally, it showed that Hst can prevent AGEs and N(ε)­carboxymethyl­lysine generation and modification of lens proteins using in vitro in human lens epithelial cell lines and ex vivo in mouse lens organ cultures. Furthermore, treatment with Hst prevented lens hardening and decreased chaperone activity in lens proteins. These results suggested that Hst and its derivatives are good candidates for the prevention of presbyopia and cataracts.

Suppression of Neuroinflammation by Coffee Component Pyrocatechol via Inhibition of NF-κB in Microglia.

According to numerous studies, it has been epidemiologically suggested that habitual coffee intake seems to prevent the onset of neurodegenerative diseases. In this study, we hypothesized that coffee consumption suppresses neuroinflammation, which is closely related to the development of neurodegenerative diseases. Using microglial BV-2 cells, we first found that the inflammatory responses induced by lipopolysaccharide (LPS) stimulation was diminished by both coffee and decaffeinated coffee through the inhibition of an inflammation-related transcription factor, nuclear factor-κB (NF-κB). Pyrocatechol, a component of roasted coffee produced by the thermal decomposition of chlorogenic acid, also exhibited anti-inflammatory activity by inhibiting the LPS-induced activation of NF-κB. Finally, in an inflammation model using mice injected with LPS into the cerebrum, we observed that intake of pyrocatechol as well as coffee decoctions drastically suppressed the accumulation of microglia and the expression of interleukin-6 (IL-6), tumor necrosis factor α (TNFα), CCL2, and CXCL1 in the inflammatory brain. These observations strongly encourage us to hypothesize that the anti-inflammatory activity of pyrocatechol as well as coffee decoction would be useful for the suppression of neurodegeneration and the prevention of the onsets of Alzheimer's (AD) and Perkinson's diseases (PD).

Regulation of the Membrane Trafficking of the Mechanosensitive Ion Channels TRPV1 and TRPV4 by Zonular Tension, Osmotic Stress and Activators in the Mouse Lens.

Lens water transport generates a hydrostatic pressure gradient that is regulated by a dual-feedback system that utilizes the mechanosensitive transient receptor potential vanilloid (TRPV) channels, TRPV1 and TRPV4, to sense changes in mechanical tension and extracellular osmolarity. Here, we investigate whether the modulation of TRPV1 or TRPV4 activity dynamically affects their membrane trafficking. Mouse lenses were incubated in either pilocarpine or tropicamide to alter zonular tension, exposed to osmotic stress, or the TRPV1 and TRPV4 activators capsaicin andGSK1016790A (GSK101), and the effect on the TRPV1 and TRPV4 membrane trafficking in peripheral fiber cells visualized using confocal microscopy. Decreases in zonular tension caused the removal of TRPV4 from the membrane of peripheral fiber cells. Hypotonic challenge had no effect on TRPV1, but increased the membrane localization of TRPV4. Hypertonic challenge caused the insertion of TRPV1 and the removal of TRPV4 from the membranes of peripheral fiber cells. Capsaicin caused an increase in TRPV4 membrane localization, but had no effect on TRPV1; while GSK101 decreased the membrane localization of TRPV4 and increased the membrane localization of TRPV1. These reciprocal changes in TRPV1/4 membrane localization are consistent with the channels acting as mechanosensitive transducers of a dual-feedback pathway that regulates lens water transport.

Capsaicin attenuates TGFß2-induced epithelial-mesenchymal-transition in lens epithelial cells in vivo and in vitro.

Posterior capsule opacification (PCO), the most common complication of cataract surgery occurring in 20-50% of patients after 2-5 years of cataract surgery, is a major problem in the aging society. The epithelial-mesenchymal transition (EMT) of lens epithelial cells after cataract surgery has been proposed as a major cause of PCO. Capsaicin, widely used as a food additive and analgesic agent, is a major pungent ingredient in red pepper. Although the effect of capsaicin on EMT has been reported in cancer cells, the biological reaction of capsaicin was unique in each cell type, and there have been no reports describing its effects on EMT earlier. In this study, we demonstrated that treatment with capsaicin inhibited TGFß2-induced EMT in vitro lens epithelial cells and ex vivo explant lens epithelial cells. Furthermore, eye drops of capsaicin inhibited the PCO model mice in vivo. Finally, we showed that capsaicin inhibited non-canonically induced Smad2/3 activation via suppression of EGFR activation and ERK phosphorylation. Our findings indicate that capsaicin and its derivatives are good candidate compounds for preventing PCO after cataract surgery.

The indole-hydantoin derivative exhibits anti-inflammatory activity by preventing the transactivation of NF-κB through the inhibition of NF-κB p65 phosphorylation at Ser276.

Indole- and hydantoin-based derivatives both exhibit anti-inflammatory activity, suggesting that the structures of indole and hydantoin are functional for this activity. In the present study, we synthesized two types of indole-hydantoin derivatives, IH-1 (5-(1H-indole-3-ylmethylene) imidazolidine-2,4-dione) and IH-2 (5-(1H-indole-3-ylmethyl) imidazolidine-2,4-dione) and examined their effects on LPS-induced inflammatory responses in murine macrophage-like RAW264.7 cells. LPS-induced inflammatory responses were not affected by indole, hydantoin, or IH-2. In contrast, IH-1 significantly inhibited the LPS-induced production of nitric oxide (NO) and secretion of CCL2 and CXCL1 by suppressing the mRNA expression of inducible NO synthase (iNOS), CCL2, and CXCL1. IH-1 markedly inhibited the LPS-induced activation of NF-κB without affecting the degradation of IκBα or nuclear translocation of NF-κB. IH-1 markedly attenuated the transcriptional activity of NF-κB by suppressing the LPS-induced phosphorylation of the NF-κB p65 subunit at Ser276. Furthermore, IH-1 prevented the LPS-induced interaction of NF-κB p65 subunit with a transcriptional coactivator, cAMP response element-binding protein (CBP). Collectively, these results revealed the potential of the novel indole-hydantoin derivative, IH-1 as an anti-inflammatory drug.

Effect of Alpha-Glucosyl-Hesperidin Consumption on Lens Sclerosis and Presbyopia.

Presbyopia is characterized by a decline in the ability to accommodate the lens. The most commonly accepted theory for the onset of presbyopia is an age-related increase in the stiffness of the lens. However, the cause of lens sclerosis remains unclear. With age, water microcirculation in the lens could change because of an increase in intracellular pressure. In the lens, the intracellular pressure is controlled by the Transient Receptor Potential Vanilloid (TRPV) 1 and TRPV4 feedback pathways. In this study, we tried to elucidate that administration of α-glucosyl-hesperidin (G-Hsd), previously reported to prevent nuclear cataract formation, affects lens elasticity and the distribution of TRPV channels and Aquaporin (AQP) channels to meet the requirement of intracellular pressure. As a result, the mouse control lens was significantly toughened compared to both the 1% and 2% G-Hsd mouse lens treatments. The anti-oxidant levels in the lens and plasma decreased with age; however, this decrease could be nullified with either 1% or 2% G-Hsd treatment in a concentration- and exposure time-dependent manner. Moreover, G-Hsd treatment affected the TRPV4 distribution, but not TRPV1, AQP0, and AQP5, in the peripheral area and could maintain intracellular pressure. These findings suggest that G-Hsd has great potential as a compound to prevent presbyopia and/or cataract formation.

Oral consumption of α-glucosyl-hesperidin could prevent lens hardening, which causes presbyopia.

Presbyopia is one of the most well-known diseases of the eye, predominantly affecting the adult population after 50 years'. Due to hardening of the lens and failure of accommodative change, patients lose the ability to focus on near objects. This eye symptom is reported to be an early symptom of age-related nuclear cataract, and we have previously reported that hesperetin treatment could delay the onset of nuclear cataractogenesis induced by sodium selenite. In this study, we examined whether oral intake of α-glucosyl-hesperidin (G-Hsd), which has greater water solubility than hesperetin, could delay the onset of presbyopia. G-Hsd treatment protected lens elasticity, upregulated the mRNA expression of anti-oxidative enzymes like glutathione reductase and thioredoxin reductase 1 in the plasma and lens, and prevented premature cataract symptoms in selenite-induced cataract rat lens. Thus, the anti-presbyopic effects of G-Hsd were attributed, at least in part, to its antioxidant effects. G-Hsd represents the first oral treatment agent with anti-presbyopia and/or anti-cataract properties.

EBP2, a novel NPM-ALK-interacting protein in the nucleolus, contributes to the proliferation of ALCL cells by regulating tumor suppressor p53.

The oncogenic fusion protein nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), found in anaplastic large-cell lymphoma (ALCL), localizes to the cytosol, nucleoplasm, and nucleolus. However, the relationship between its localization and transforming activity remains unclear. We herein demonstrated that NPM-ALK localized to the nucleolus by binding to nucleophosmin 1 (NPM1), a nucleolar protein that exhibits shuttling activity between the nucleolus and cytoplasm, in a manner that was dependent on its kinase activity. In the nucleolus, NPM-ALK interacted with Epstein-Barr virus nuclear antigen 1-binding protein 2 (EBP2), which is involved in rRNA biosynthesis. Moreover, enforced expression of NPM-ALK induced tyrosine phosphorylation of EBP2. Knockdown of EBP2 promoted the activation of the tumor suppressor p53, leading to G0 /G1 -phase cell cycle arrest in Ba/F3 cells transformed by NPM-ALK and ALCL patient-derived Ki-JK cells, but not ALCL patient-derived SUDH-L1 cells harboring p53 gene mutation. In Ba/F3 cells transformed by NPM-ALK and Ki-JK cells, p53 activation induced by knockdown of EBP2 was significantly inhibited by Akt inhibitor GDC-0068, mTORC1 inhibitor rapamycin, and knockdown of Raptor, an essential component of mTORC1. These results suggest that the knockdown of EBP2 triggered p53 activation through the Akt-mTORC1 pathway in NPM-ALK-positive cells. Collectively, the present results revealed the critical repressive mechanism of p53 activity by EBP2 and provide a novel therapeutic strategy for the treatment of ALCL.

Degradation of connexin 50 protein causes waterclefts in human lens.

Cataracts are mainly classified into three types: cortical cataracts, nuclear cataracts, and posterior subcapsular cataracts. In addition, retrodots and waterclefts are cataract subtypes that cause decreased visual function. To maintain an orderly and tightly packed arrangement to minimize light scattering, adhesion molecules such as connexins and aquaporin 0 (AQP0) are highly expressed in the lens. We hypothesized that some main and/or subcataract type(s) are correlated with adhesion molecule degradation. Lens samples were collected from cataract patients during cataract surgery, and mRNA and protein expression levels were measured by real-time RT-PCR and western blotting, respectively. The mRNA levels of adhesion molecules were not significantly different among any cataract types. Moreover, AQP0 and connexin 46 protein expressions were unchanged among patients. However, connexin 50 protein level was significantly decreased in the lens of patients with WC cataract subtype. P62 and LC3B proteins were detected in the WC patients' lenses, but not in other patients' lenses. These results suggest that more research is needed on the subtypes of cataracts besides the three major types of cataract for tailor-made cataract therapy.

[Molecular Basis of Preventive Effects of Habitual Coffee Intake against Chronic Diseases].

Epidemiological studies have shown that coffee consumption may be associated with a lower risk of developing several chronic disorders. To elucidate the molecular mechanism of the effects of coffee, we analyzed molecular response upon exposure to coffee extract using cellular and animal models of these diseases. As obesity is recognized as a major risk factor for these chronic diseases, we investigated the effect of coffee on adipogenesis using mouse preadipocyte 3T3-L1 cells. We found that coffee induced proteasomal degradation of IRS-1, leading to reduction of PPARγ expression, a master transcription factor for adipogenesis. Reduction in weight as well as in IRS-1 expression was detected in the fat tissues of the high fat-diet-fed mice when reared with 60% coffee for 7 weeks. As for Alzheimer's disease, we analyzed the effect of coffee on amyloid ß (Aß) production in human neuronal SH-SY5Y cells. We found a 20% reduction in Aß production when treated with 2.5% coffee for 2 d. This reduction was due to proteasomal degradation of BACE1 (ß-secretase), which was activated by protein kinase A. In addition, coffee ameliorates LPS-induced inflammatory responses in RAW264.7 macrophages by reducing NFκB activity and Nrf2 activation. Roasted coffee prevents selenite-induced cataractogenesis by ameliorating antioxidant loss. Pyrocatechol, a component of roasted coffee, also reduced Aß production and exhibits anti-inflammatory effects by a similar mechanism as coffee. Our results suggest that roasting coffee beans to generate pyrocatechol is necessary for the preventive effects of coffee intake on the chronic diseases.

Coffee decoction enhances tamoxifen proapoptotic activity on MCF-7 cells.

The consumption of coffee has been suggested to effectively enhance the therapeutic effects of tamoxifen against breast cancer; however, the underlying molecular mechanisms remain unclear. We herein attempted to clarify how coffee decoction exerts anti-cancer effects in cooperation with tamoxifen using the estrogen receptor α (ERα)-positive breast cancer cell line, MCF-7. The results obtained showed that coffee decoction down-regulated the expression of ERα, which was attributed to caffeine inhibiting its transcription. Coffee decoction cooperated with tamoxifen to induce cell-cycle arrest and apoptotic cell death, which may have been mediated by decreases in cyclin D1 expression and the activation of p53 tumor suppressor. The inclusion of caffeine in coffee decoction was essential, but not sufficient, to induce cell-cycle arrest and apoptotic cell death, suggesting the requirement of unknown compound(s) in coffee decoction to decrease cyclin D1 expression and activate apoptotic signaling cascades including p53. The activation of p53 through the cooperative effects of these unidentified component(s), caffeine, and tamoxifen appeared to be due to the suppression of the ERK and Akt pathways. Although the mechanisms by which the suppression of these pathways induces p53-mediated apoptotic cell death remain unclear, the combination of decaffeinated coffee, caffeine, and tamoxifen also caused cell-cycle arrest and apoptotic cell death, suggesting that unknown compound(s) present in decaffeinated coffee cooperate with caffeine and tamoxifen.

Kampo medicines, Rokumigan, Hachimijiogan, and Goshajinkigan, significantly inhibit glucagon-induced CREB activation.

The pathophysiology of type 2 diabetes mellitus (T2DM) is characterized by not only insulin resistance, but also the abnormal regulation of glucagon secretion, suggesting that antagonizing the glucagon-induced signaling pathway has therapeutic potential in the treatment of T2DM. Although various Kampo medicines (traditional herbal medicines) are often utilized to ameliorate the symptoms of T2DM, their effects on glucagon signaling have not yet been clarified. In the present study, we examined the effects of nine types of representative Kampo formulations prescribed for T2DM on glucagon-induced CREB activation in HEK293T cells stably expressing glucagon receptor (Gcgr) and a hepatic cell line HepG2. Among these Kampo medicines, Rokumigan, Hachimijiogan, and Goshajinkigan significantly suppressed the glucagon-induced transactivation of the cAMP-responsive element (CRE)-binding protein (CREB) by inhibiting its interaction with CREB-binding protein (CBP), which led to a reduction in the expression of phosphoenolpyruvate carboxykinase (PEPCK) mRNA. Furthermore, among the crude drugs commonly contained in these three Kampo medicines, Rehmannia Root (Jio), Moutan Bark (Botampi), and Cornus Fruit (Shanzhuyu) exerted inhibitory effects on glucagon-induced CREB activation. Collectively, the present results provide a novel mechanism, the inhibition of glucagon signaling, by which Rokumigan, Hachimijiogan, and Goshajinkigan improve the symptoms of T2DM.

Pyrocatechol, a component of coffee, suppresses LPS-induced inflammatory responses by inhibiting NF-κB and activating Nrf2.

Coffee is a complex mixture of many bioactive compounds possessing anti-inflammatory properties. However, the mechanisms by which coffee exerts anti-inflammatory effects remains unclear and the active ingredients have not yet been identified. In this study, we found that coffee extract at more than 2.5%(v/v) significantly inhibited LPS-induced inflammatory responses in RAW264.7 cells and that anti-inflammatory activity of coffee required the roasting process. Interestingly, we identified pyrocatechol, a degradation product derived from chlorogenic acid during roasting, as the active ingredient exhibiting anti-inflammatory activity in coffee. HPLC analysis showed that 124 µM pyrocatechol was included in 100% (v/v) roasted coffee. A treatment with 5%(v/v) coffee extract and more than 2.5 µM pyrocatechol inhibited the LPS-induced activation of NF-κB and also significantly activated Nrf2, which acts as a negative regulator in LPS-induced inflammation. Furthermore, intake of 60% (v/v) coffee extract and 74.4 µM pyrocatechol, which is the concentration equal to contained in 60% (v/v) coffee, markedly inhibited the LPS-induced inflammatory responses in mice. Collectively, these results demonstrated that pyrocatechol, which was formed by the roasting of coffee green beans, is one of the ingredients contributing to the anti-inflammatory activity of coffee.

Oral intake of α­glucosyl­hesperidin ameliorates selenite­induced cataract formation.

Hesperetin is a natural flavonoid with robust antioxidant properties. Our previous study reported that hesperetin can prevent cataract formation. However, an important consideration regarding hesperetin consumption is the limited bioavailability due to its poor solubility. The present study investigated the anti­cataract effects of α­glucosyl hesperidin in vivo and in vitro using a selenite­induced cataract model. SD rats (age, 13 days) were orally administered PBS (0.2 ml) or α­glucosyl hesperidin (200 mg/kg) on days 0, 1 and 2. Sodium selenite was subcutaneously administered to the rats 4 h after the first oral administration on day 0. Antioxidant levels in the lens and blood were measured on day 6. In vitro, human lens epithelial cells were treated with sodium selenite (10 µM) and/or hesperetin (50 or 100 mM) for 24 h and analyzed for apoptosis markers using sub­G1 population and Annexin V­FITC/propidium iodide staining and DNA ladder formation. α­glucosyl hesperidin treatment significantly reduced the severity of selenite­induced cataract. The level of antioxidants was significantly reduced in the selenite­treated rats compared with in the controls; however, they were normalized with α­glucosyl hesperidin treatment. In vitro, hesperetin could significantly reduce the number of cells undergoing apoptosis induced by sodium selenite in human lens epithelial cell lines. Overall, oral consumption of α­glucosyl hesperidin could delay the onset of selenite­induced cataract, at least in part by modulating the selenite­induced cell death in lens epithelial cells.

N-Acetyl cysteine prevents activities of STAT3 inhibitors, Stattic and BP-1-102 independently of its antioxidant properties.

BACKGROUND: Inhibitors for signal transducer and activator of transcription 3 (STAT3), Stattic, BP-1-102, and LLL12 significantly induce apoptosis in transformed Ba/F3 cells expressing an oncogenic fusion protein, nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) that induces the activation of STAT3. We found that the antioxidant reagent, N-acetyl cysteine (NAC) prevented the abilities of Stattic and BP-1-102, but not LLL12 to induce apoptosis in transformed cells expressing NPM-ALK, providing a novel problem in use of STAT3 inhibitors. We herein investigated the mechanisms how NAC prevented the effects of Sttatic and BP-1-102. METHODS: Ba/F3 cells expressing NPM-ALK and SUDHL-1 cells were treated with antioxidants such as NAC, Trolox or edaravone in combination with STAT3 inhibitors. Phosphorylation of STAT3, cell proliferation rate, cell viability, cell cycle, internucleosomal DNA fragmentation and the intracellular accumulation of reactive oxygen species (ROS) was investigated. The binding of STAT3 inhibitors and NAC was analyzed by LC-MS. RESULTS: NAC but not Trolox and edaravone diminished the abilities of Stattic and BP-1-102 to induce apoptosis in cells expressing NPM-ALK. The ROS levels in cells expressing NPM-ALK were not markedly affected by the treatments with Stattic and BP-1-102 in combination with NAC, suggesting that NAC inhibited the activity of Stattic and BP-1-102 independent of its antioxidant activity. LC-MS analysis revealed that NAC directly bound to Stattic and BP-1-102. Furthermore, these NAC adducts exhibited no cytotoxicity, and failed to affect the activity of STAT3. CONCLUSIONS: NAC antagonizes the activities of Stattic and BP-1-102, which inhibit STAT3 activation by interacting with cysteine residues in STAT3.

Methotrexate significantly induces apoptosis by inhibiting STAT3 activation in NPM-ALK-positive ALCL cells.

Anaplastic large cell lymphoma (ALCL) is associated with a characteristic chromosomal translocation that generates the oncogenic fusion protein, nucleophosmin-anaplastic lymphoma kinase (NPM-ALK). Methotrexate is a commonly used chemotherapeutic drug in the treatment of multiple cancers due to its inhibition of dihydrofolate reductase (DHFR), which suppresses the synthesis of DNA. In the present study, we found that low-dose methotrexate significantly induced apoptosis in transformed Ba/F3 cells expressing NPM-ALK by inhibiting the activation of signal transducer and activator of transcription factor 3 (STAT3), a critical downstream molecule of NPM-ALK. Although methotrexate prevented the phosphorylation of STAT3, it did not affect the activity of NPM-ALK. A co-treatment with folinic acid prevented the methotrexate-induced inhibition of STAT3 activation and induction of apoptosis, suggesting that methotrexate exerts its cytotoxic effects by depleting tetrahydrofolate (THF) in transformed cells by NPM-ALK. Furthermore, methotrexate induced the down-regulation of the anti-apoptotic protein, MCL-1, DNA damage, and the activation of a p53 tumor suppressor, leading to apoptosis through the inhibition of STAT3. Methotrexate significantly induced apoptosis in ALK inhibitor-resistant cells expressing the NPM-ALK mutant harboring the point mutation, G262R, and in ALCL patient-derived NPM-ALK-positive Ki-JK cells. Collectively, these results demonstrate the potential therapeutic application of methotrexate, which inhibits the activation of STAT3, to NPM-ALK-positive ALCL.

Tyrosine-phosphorylated SOCS3 negatively regulates cellular transformation mediated by the myeloproliferative neoplasm-associated JAK2 V617F mutant.

In the majority of myeloproliferative neoplasms (MPNs) patients, a point mutation, V617F has been found in Janus kinase 2 (JAK2) gene, and this JAK2 mutant provoked aberrant signaling pathway. In the current study, we found that suppressor of cytokine signaling proteins 3 (SOCS3) possessed the tumor suppressive activity against the JAK2 V617F mutant-provoked cellular transformation. The knockdown of SOCS3 increased the expression level of the JAK2 V617F mutant, which enhanced the activation of signaling mediators, including signal transducer and activator of transcription 3 and 5 (STAT3, STAT5) and extracellular signal-regulated kinase (ERK), and also increased of the proliferation rate and tumorigenesis activity of Ba/F3 cells expressing the JAK2 V617F mutant and erythropoietin receptor (EpoR). In contrast, the enforced expression of SOCS3 significantly inhibited the JAK2 V617F mutant-induced activation of downstream signaling molecules, cell proliferation, and tumorigenesis by down-regulating the expression level of the JAK2 V617F mutant. SOCS3 interacted with the JAK2V617F mutant through its SH2 domain and was phosphorylated at Tyr-204 and Tyr-221 in its SOCS box by the JAK2V617F mutant. SOCS3 mutants carrying a mutation in the SH2 domain (R71E) and a substitution at Tyr-221 (Y221F) failed to exert inhibitory effects on JAK2V617F mutant-induced cellular transformation and tumorigenesis. Collectively, these results imply that SOCS3 plays a negative role in the JAK2 V617F mutant-induced oncogenic signaling pathway through its SH2 domain and the phosphorylation of Tyr-221 in its SOCS box.

Coffee brew intake can prevent the reduction of lens glutathione and ascorbic acid levels in HFD-fed animals.

The lens has high concentrations of glutathione (GSH) and ascorbic acid (AsA) to maintain redox activity and prevent cataract formation, which is the leading cause of visual impairment worldwide. Metabolic syndrome is reported to be linked with a higher risk of age-associated cataract. As it was demonstrated previously that coffee consumption improved high-fat diet (HFD) -induced metabolic symptoms, it was hypothesized that coffee intake could delay the onset of obesity related-cataract; however, the effect of coffee consumption on this type of cataract remains unknown. Four-week-old male C57BL/6JJms SLC mice were divided into two groups and were provided ad libitum access to either a control diet (control groups) or a HFD (HFD groups). The control groups and HFD groups were further divided into three or four subgroups for each experiment. Coffee intake markedly reduced the increase in body weight in a roasting-time and concentration-dependent manner. Coffee consumption also prevented the HFD-induced decrease in the concentration of GSH and AsA, and treatment with pyrocatechol or caffeine also restored the reduction of antioxidant compounds. Plasma cholesterol and triglycerides were significantly higher in HFD groups; however, coffee brew or coffee constituent treatment in the HFD-fed mice group prevented elevation of these levels. Caffeine is a major coffee component and pyrocatechol is generated thought the roasting process. These results revealed that caffeine and pyrocatechol in coffee brew may be the key constituents responsible for preventing the reduction of lens GSH and AsA in HFD-fed animals.

Chemoenzymatic synthesis of hydroxytyrosol monoesters and their suppression effect on nitric oxide production stimulated by lipopolysaccharides.

Fatty acid monoesters of hydroxytyrosol [2-(3,4-dihydroxyphenyl)ethanol] were synthesized in two steps from tyrosol (4-hydroxyphenylethanol) by successive Candida antarctica lipase B-catalyzed chemoselective acylation on the primary aliphatic hydroxy group over phenolic hydroxy group in tyrosol, and 2-iodoxybenzoic acid (IBX)-mediated hydroxylation adjacent to the remaining free phenolic hydroxy group. Examination of their suppression effects on nitric oxide production stimulated by lipopolysaccharides in RAW264.7 cells showed that hydroxytyrosol butyrate exhibited the highest inhibition (IC50 7.0 µM) among the tested compounds.