Effects of a Tea Cultivar "MK5601" on Behaviors and Hippocampal Neurotrophin-3 Levels in Middle-Aged Mice.

Dietary factors are thought to play an important role in the prevention of cognition diseases and depression in late life. In the present study, we compared the effects between the theogallin-rich tea cultivar, "MK5601" and a common Japanese tea cultivar, "Yabukita" on behaviors and hippocampal neurotrophin levels in experimental animals. Middle-aged mice (aged 8 mo) were given either of the tea infusions or water ad libitum for 4 mo. In the novel object location test, the middle-aged mice drinking water or "Yabukita" performed worse than young mice (aged 2-3 mo) although the middle-aged mice drinking "MK5601" retained spatial memory at the same level as the young mice. We also found that the middle-aged mice drinking "MK5601" showed high levels of neurotrophin-3 in the hippocampus. In conclusion, the "MK5601" tea infusion appears to be effective in preventing age-related changes in cognitive function, as compared with a common Japanese tea cultivar.

Plasma Homocysteine Concentration is Associated with the Expression Level of Folate Receptor 3.

Folic acid and folate receptors (FOLRs) play an important role in the downregulation of homocysteine (Hcy), a risk factor of Alzheimer's disease, thrombosis, neuropsychiatric illness and fractures. While several studies have reported that FOLR1 and FOLR2 import folic acid into cells, the role of FOLR3 remains unknown. In this study, we evaluated the impact of FOLR3 on the metabolism of Hcy alongside its protective effect against homocysteine-induced neurotoxicity. To reveal the role of FOLR3, we constructed FOLR3-overexpressed HEK293 cells (FOLR3+ cells) and evaluated cell growth, folic acid intake and Hcy-induced neurotoxicity. Subjects with a high expression of FOLR3 exhibited low levels of plasma homocysteine. The ectopic expression of FOLR3 enhanced cell growth, and the enhanced effect was neutralised by folic acid-deficient media. The Western blot analysis revealed that FOLR3 is secreted into cell supernatant. The folic acid intake of FOLR3+ cells was higher than that of wild-type cells. Supernatant from FOLR3+ cells showed a protective effect on Hcy-induced cytotoxicity. FOLR3 expression in plasma is negatively correlated with plasma homocysteine. Our study emphasizes the role of FOLR3 in the intake of folic acid into cells on the one hand and its protective role in Hcy-induced cytotoxicity on the other.

Saturated fatty acid attenuates anti-obesity effect of green tea.

Green tea and its major polyphenol epigallocatechin-3-O-gallate (EGCG) have suppressive effect on dietary obesity. However, it remains unsolved what type of diet on which they exhibit high or low anti-obesity effect. In the present study, we investigated whether anti-obesity effect of green tea differs depending on composition of fats or fatty acids that consist high-fat (HF) diet in mouse model. Green tea extract (GTE) intake dramatically suppressed weight gain and fat accumulation induced by olive oil-based HF diet, whereas the effects on those induced by beef tallow-based HF diet were weak. GTE also effectively suppressed obesity induced by unsaturated fatty acid-enriched HF diet with the stronger effect compared with that induced by saturated fatty acid-enriched HF diet. These differences would be associated with the increasing action of GTE on expression of PPARδ signaling pathway-related genes in the white adipose tissue. Expressions of genes relating to EGCG signaling pathway that is critical for exhibition of physiological effects of EGCG were also associated with the different effects of GTE. Here, we show that anti-obesity effect of GTE differs depending on types of fats or fatty acids that consist HF diet and could be attenuated by saturated fatty acid.

Green Tea Polyphenol EGCG Upregulates Tollip Expression by Suppressing Elf-1 Expression.

TLR signaling is critical to innate immune system regulation; however, aberrant TLR signaling is involved in several diseases, including insulin resistance, Alzheimer's disease, and tumor metastasis. Moreover, a recent study found that TLR-4 signaling pathway inhibition might be a target for the suppression of chronic inflammatory disorders. In this article, we show that the green tea polyphenol epigallocatechin-3-O-gallate (EGCG) increases the expression of Toll interacting protein, a strong inhibitor of TLR4 signaling, by suppressing the expression of E74-like ETS transcription factor 1 (Elf-1). A mechanistic study revealed that EGCG suppressed Elf-1 expression via protein phosphatase 2A/cyclic GMP (cGMP)-dependent mechanisms. We also confirmed that orally administered EGCG and a cGMP inducer upregulated Toll interacting protein expression, increased intracellular levels of cGMP in macrophages, and suppressed Elf-1 expression. These data support EGCG and a cGMP inducer as potential candidate suppressors of TLR4 signaling.

Hydrogen sulphide donors selectively potentiate a green tea polyphenol EGCG-induced apoptosis of multiple myeloma cells.

Hydrogen sulphide (H2S) is a colourless gas with the odour of rotten eggs and has recently been recognized as a signal mediator in physiological activities related with the regulation of homeostasis, the vascular system and the inflammatory system. Here we show that H2S donors, including sodium hydrogen sulphide (NaHS), GYY 4137 and diallyltrisulfide (DATS), synergistically enhanced the anti-cancer effect of a green tea polyphenol (-)-epigallocatechin-3-O-gallate (EGCG) against multiple myeloma cells without affecting normal cells. NaHS significantly potentiated the anti-cancer effect of EGCG and prolonged survival in a mouse xenograft model. In this mechanism, H2S enhanced apoptotic cell death through cyclic guanosine monophosphate (cGMP)/acid sphingomyelinase pathway induced by EGCG. Moreover, NaHS reduced the enzyme activity of cyclic nucleotide phosphodiesterase that is known as cGMP negative regulator. In conclusion, we identified H2S as a gasotransmitter that potentiates EGCG-induced cancer cell death.

Green Tea Polyphenol Epigallocatechin-3-gallate Suppresses Toll-like Receptor 4 Expression via Up-regulation of E3 Ubiquitin-protein Ligase RNF216.

Toll-like receptor 4 (TLR4) plays an essential role in innate immunity through inflammatory cytokine induction. Recent studies demonstrated that the abnormal activation of TLR4 has a pivotal role in obesity-induced inflammation, which is associated with several diseases, including hyperinsulinemia, hypertriglyceridemia, and cardiovascular disease. Here we demonstrate that (-)-epigallocatechin-3-O-gallate, a natural agonist of the 67-kDa laminin receptor (67LR), suppressed TLR4 expression through E3 ubiquitin-protein ring finger protein 216 (RNF216) up-regulation. Our data indicate cyclic GMP mediates 67LR agonist-dependent RNF216 up-regulation. Moreover, we show that the highly absorbent 67LR agonist (-)-epigallocatechin-3-O-(3-O-methyl)-gallate (EGCG3″Me) significantly attenuated TLR4 expression in the adipose tissue. EGCG3″Me completely inhibited the high-fat/high-sucrose (HF/HS)-induced up-regulation of tumor necrosis factor α in adipose tissue and serum monocyte chemoattractant protein-1 increase. Furthermore, this agonist intake prevented HF/HS-induced hyperinsulinemia and hypertriglyceridemia. Taken together, 67LR presents an attractive target for the relief of obesity-induced inflammation.

Oligomer formation of a tea polyphenol, EGCG, on its sensing molecule 67 kDa laminin receptor.

Green tea polyphenol (-)-epigallocatechin-3-O-gallate (EGCG) has been attributed to the activation of its cell surface sensing receptor 67 kDa laminin receptor (67LR). However, the action of EGCG to activate 67LR remains unknown. Here we show that EGCG undergoes oligomer formation on its surface receptor 67LR.

The Combination of Green Tea Extract and Eriodictyol Inhibited High-Fat/High-Sucrose Diet-Induced Cholesterol Upregulation Is Accompanied by Suppression of Cholesterol Synthesis Enzymes.

Western diets induce obesity associated with an increased risk of hypercholesterolaemia. Indeed, obesity-induced hypercholesterolaemia is correlated with increased coronary cardiovascular disease (CVD) risk. Male C57BL/6J mice were fed a normal diet, high-fat and high-sucrose diet (HF/HS), HF/HS with green tea extract powder diet (HF/HS+GT), HF/HS with eriodictyol diet (HF/HS+Eri), or HF/HS with green tea extract powder and eriodictyol diet (HF/HS+GT+Eri) for 8 wk. Body weight was lower in the HF/HS+GT+Eri group than in the HF/HS group (-8.3%, p<0.01). The HF/HS diet elicited an upregulation of total cholesterol levels (-63%, p<0.001), and low-density lipoprotein (LDL) levels (-89%, p<0.001) were significantly suppressed by the GT+Eri diet. Conversely, no change (p>0.05) was observed in the HF/HS+GT and HF/HS+Eri groups. The HF/HS diet-induced hepatic mRNA increase in 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) was ameliorated (-73%) by the oral administration of green tea extract and eriodictyol. Moreover, the GT+Eri diet suppressed HF/HS diet-induced upregulation of 3-hydroxy-3-methylglutaryl-coenzyme A synthase (HMGCS) (-75%, p<0.05). Furthermore, the LDL receptor (LDLR) levels were higher in the HF/HS+GT+Eri group (+50%, p<0.05) than in the HF/HS group. These results suggest that a combination of green tea and eriodictyol decreases cholesterol levels, particularly LDL levels, accompanied by the suppression of HMGCR and HMGCS levels and upregulation of LDLR levels in the liver.

Equol suppresses inflammatory response and bone erosion due to rheumatoid arthritis in mice.

Rheumatoid arthritis (RA) is a chronic and systemic autoimmune inflammatory disease. Typical pathological findings of RA include persistent synovitis and bone degradation in the peripheral joints. Equol, a metabolite of the major soybean isoflavone daidzein, shows superior bioactivity than other isoflavones. We investigated the effects of equol administration on inflammatory response and bone erosion in mice with collagen-induced arthritis (CIA). The severity of arthritis symptoms was significantly low in the equol-administered CIA mice. In addition, equol administration improved the CIA-induced bone mineral density decline. In the inflamed area of CIA mice, equol administration suppressed the expression of interleukin-6 and its receptor. Furthermore, equol reduced the expression of genes associated with bone formation inhibition, osteoclast and immature osteoblast specificity and cartilage destruction. These results suggest that equol suppresses RA development and RA-induced bone erosion by regulating inflammation and bone metabolism.

Green Tea Catechin Metabolites Exert Immunoregulatory Effects on CD4(+) T Cell and Natural Killer Cell Activities.

Tea catechins, such as (-)-epigallocatechin-3-O-gallate (EGCG), have been shown to effectively enhance immune activity and prevent cancer, although the underlying mechanism is unclear. Green tea catechins are instead converted to catechin metabolites in the intestine. Here, we show that these green tea catechin metabolites enhance CD4(+) T cell activity as well as natural killer (NK) cell activity. Our data suggest that the absence of a 4'-hydroxyl on this phenyl group (B ring) is important for the effect on immune activity. In particular, 5-(3',5'-dihydroxyphenyl)-γ-valerolactone (EGC-M5), a major metabolite of EGCG, not only increased the activity of CD4(+) T cells but also enhanced the cytotoxic activity of NK cells in vivo. These data suggest that EGC-M5 might show immunostimulatory activity.

Green tea polyphenol epigallocatechin-O-gallate induces cell death by acid sphingomyelinase activation in chronic myeloid leukemia cells.

An epidemiological study showed that green tea consumption is associated with a reduced risk of hematopoietic malignancy. The major green tea polyphenol epigallocatechin­3-O-gallate (EGCG) is reported to have anticancer effects. Chronic myeloid leukemia (CML) is a major hematopoietic malignancy characterized by expansion of myeloid cells. In the present study, we showed EGCG-induced acid sphingomyelinase (ASM) activation and lipid raft clustering in CML cells. The ASM inhibitor desipramine significantly reduced EGCG-induced cell death. Protein kinase Cδ is a well­known kinase that plays an important role in ASM activation. We observed EGCG-induced phosphorylation of protein kinase Cδ at Ser664. Importantly, EGCG-induced ASM activation was significantly reduced by pretreatment of CML cells with the soluble guanylate cyclase inhibitor NS2028, suggesting that EGCG induced ASM activation through the cyclic guanosine monophosphate (cGMP)-dependent pathway. Indeed, pharmacological inhibition of a cGMP-negative regulator enhanced the anti-CML effect of EGCG. These results indicate that EGCG-induced cell death via the cGMP/ASM pathway in CML cells.

Metabolic profiling-based data-mining for an effective chemical combination to induce apoptosis of cancer cells.

Green tea extract (GTE) induces apoptosis of cancer cells without adversely affecting normal cells. Several clinical trials reported that GTE was well tolerated and had potential anti-cancer efficacy. Epigallocatechin-3-O-gallate (EGCG) is the primary compound responsible for the anti-cancer effect of GTE; however, the effect of EGCG alone is limited. To identify GTE compounds capable of potentiating EGCG bioactivity, we performed metabolic profiling of 43 green tea cultivar panels by liquid chromatography-mass spectrometry (LC-MS). Here, we revealed the polyphenol eriodictyol significantly potentiated apoptosis induction by EGCG in vitro and in a mouse tumour model by amplifying EGCG-induced activation of the 67-kDa laminin receptor (67LR)/protein kinase B/endothelial nitric oxide synthase/protein kinase C delta/acid sphingomyelinase signalling pathway. Our results show that metabolic profiling is an effective chemical-mining approach for identifying botanical drugs with therapeutic potential against multiple myeloma. Metabolic profiling-based data mining could be an efficient strategy for screening additional bioactive compounds and identifying effective chemical combinations.

In situ label-free visualization of orally dosed strictinin within mouse kidney by MALDI-MS imaging.

Matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI) is a powerful technique for visualizing the distribution of a wide range of biomolecules within tissue sections. However, methodology for visualizing a bioactive ellagitannin has not yet been established. This paper presents a novel in situ label-free MALDI-MSI technique for visualizing the distribution of strictinin, a bioactive ellagitannin found in green tea, within mammalian kidney after oral dosing. Among nine representative matrix candidates, 1,5-diaminonaphthalene (1,5-DAN), harmane, and ferulic acid showed higher sensitivity to strictinin spotted onto a MALDI sample plate. Of these, 1,5-DAN enables visualization of a two-dimensional image of strictinin directly spotted on mouse kidney sections with the highest sensitivity. Furthermore, 1,5-DAN-based MALDI-MSI could detect the unique distribution of orally dosed strictinin within kidney sections. This in situ label-free imaging technique will contribute to the localization analysis of strictinin and its biological mechanisms.

Green tea extract containing a highly absorbent catechin prevents diet-induced lipid metabolism disorder.

We investigated the effects of extracts of Benifuuki (a tea cultivar that contains methylated catechins such as epigallocatechin-3-O-(3-O-methyl) gallate (EGCG3"Me)) in mice fed a high-fat/high-sucrose (HF/HS) diet. This tea cultivar was then compared with an extract of Yabukita (a popular tea cultivar that lacks methylated catechins). For 6 weeks, C57BL/6J mice were fed either HF/HS diet with or without tea extracts from tea cultivars, which contained almost identical ingredients except for methylated catechins (i.e., Yabukita (0.2% and 1%) or Benifuuki (0.2% and 1%) extract powders). Supplementation with Benifuuki 0.2% markedly lowered plasma levels of TG and NEFAs compared with mice supplemented with Yabukita 0.2%. The diet containing Benifuuki 1% decreased adipose tissue weights, liver TG, and expression of lipogenic genes in the liver. These results suggested that Benifuuki had much greater lipid-lowering effects than Yabukita. Taken together, these data suggest that methylated catechins direct the strong lipid-lowering activity of Benifuuki.

Green tea polyphenol EGCG induces lipid-raft clustering and apoptotic cell death by activating protein kinase Cδ and acid sphingomyelinase through a 67 kDa laminin receptor in multiple myeloma cells.

EGCG [(-)-epigallocatechin-3-O-gallate], the major polyphenol of green tea, has cancer chemopreventive and chemotherapeutic activities. EGCG selectively inhibits cell growth and induces apoptosis in cancer cells without adversely affecting normal cells; however, the underlying molecular mechanism in vivo is unclear. In the present study, we show that EGCG-induced apoptotic activity is attributed to a lipid-raft clustering mediated through 67LR (67 kDa laminin receptor) that is significantly elevated in MM (multiple myeloma) cells relative to normal peripheral blood mononuclear cells, and that aSMase (acid sphingomyelinase) is critical for the lipid-raft clustering and the apoptotic cell death induced by EGCG. We also found that EGCG induces aSMase translocation to the plasma membrane and PKCδ (protein kinase Cδ) phosphorylation at Ser664, which was necessary for aSMase/ceramide signalling via 67LR. Additionally, orally administered EGCG activated PKCδ and aSMase in a murine MM xenograft model. These results elucidate a novel cell-death pathway triggered by EGCG for the specific killing of MM cells.