Preventive effects of tea and tea catechins against influenza and acute upper respiratory tract infections: a systematic review and meta-analysis.

PURPOSE: Gargling with tea has protective effects against influenza infection and upper respiratory tract infection (URTI). To evaluate if tea and tea catechin consumption has the same protective effects as gargling with tea, we performed a systematic review and meta-analysis. METHODS: We performed a comprehensive literature search using the PubMed, Cochrane Library, Web of Science, and Ichu-shi Web databases. The search provided six randomized controlled trials (RCTs) and four prospective cohort studies (n = 3748). The quality of each trial or study was evaluated according to the Cochrane risk-of-bias tool or Newcastle-Ottawa Scale. We collected data from publications meeting the search criteria and conducted a meta-analysis of the effect of tea gargling and tea catechin consumption for preventing URTI using a random effects model. RESULTS: Tea gargling and tea catechin consumption had significant preventive effects against URTI (risk ratio [RR] = 0.74, 95% confidence interval [CI] 0.64-0.87). In sub-analyses, a significant preventive effect was observed by study type (prospective cohort study: RR = 0.67, 95% CI 0.50-0.91; RCT: RR = 0.79, 95% CI 0.66-0.94) and disease type (influenza: RR = 0.69, 95% CI 0.58-0.84; acute URTI: RR = 0.78, 95% CI 0.62-0.98). Both gargling with tea and consuming tea catechins effectively protected against URTI (tea and tea catechins consumption: RR = 0.68, 95% CI 0.52-0.87; tea gargling: RR = 0.83, 95% CI 0.72-0.96). CONCLUSION: Our findings suggest that tea gargling and tea catechin consumption may have preventive effects against influenza infection and URTI. The potential effectiveness of these actions as non-pharmaceutical interventions, however, requires further investigation.

Green tea extracts reduce leukocyte cell-Derived chemotaxin 2 and selenoprotein P levels in the livers of C57BL/6J mice fed a high-fat diet.

Epidemiological studies suggest that green tea extracts (GTEs), including catechins such as epigallocatechin gallate and epicatechin gallate, have a beneficial effect on obesity, hyperglycemia, insulin resistance, endothelial dysfunction, and inflammation. Although several studies have shown that catechins directly modulate the cellular and molecular alterations in the liver tissue, the contributions of indirect mechanisms underlying these systemic effects of catechins remain unclear. In this study, we report that, in the C57BL/6J mouse liver, GTEs reduce high-fat diet-induced increases in the levels of hepatokines, liver-derived secretary proteins such as leukocyte cell-derived chemotaxin 2 and selenoprotein P production, which have been shown to induce systemic adverse effects, including several metabolic diseases. These findings suggest that the systemic effects of GTEs involve the regulation of hepatokine production as an indirect mechanism.

Tyrosine Is a Booster of Leucine-Induced Muscle Anabolic Response.

Leucine (Leu), an essential amino acid, is known to stimulate protein synthesis in the skeletal muscle via mTOR complex 1 (mTORC1) activation. However, the intrinsic contribution of other amino acids to Leu-mediated activation of mTORC1 signaling remains unexplored. This study aimed to identify amino acids that can promote mTORC1 activity in combination with Leu and to assess the effectiveness of these combinations in vitro and in vivo. We found that tyrosine (Tyr) enhanced Leu-induced phosphorylation of S6 kinase (S6K), an indicator of mTORC1 activity, although it exerted no such effect individually. This booster effect was observed in C2C12 cells, isolated murine muscle, and the skeletal muscles of mice orally administered the amino acids. To explore the molecular mechanisms underlying this Tyr-mediated booster effect, the expression of the intracellular Leu sensors, Sestrin1 and 2, was suppressed, and the cells were treated with Leu and Tyr. This suppression enabled Tyr alone to induce S6K phosphorylation and enhanced the booster effect, suggesting that Tyr possibly contributes to mTORC1 activation when Sestrin-GAP activity toward Rags 2 (GATOR2) is dissociated through Sestrin knockdown or the binding of Sestrins to Leu. Collectively, these results indicate that Tyr is a key regulator of Leu-mediated protein synthesis.

Protein-bound sialic acid in saliva contributes directly to salivary anti-influenza virus activity.

The oral cavity is an entrance for respiratory viruses, such as influenza. Recently, saliva has been shown to exert both antimicrobial and antiviral activities. Thus, saliva may be a biological factor that contributes to the prevention of influenza infection. However, the actual salivary anti-influenza A virus (IAV) activity in individuals and its determinant factors are unknown. By assessing individual variations in salivary anti-IAV activity in 92 people using an established new high-throughput system in this study, we found that the anti-IAV activity varied widely between individuals and showed a significant positive correlation with protein-bound sialic acid (BSA) level (ρ = 0.473; p < 0.001). Furthermore, the anti-IAV activity of saliva with enzymatically reduced BSA content was significantly lower. These results indicate that BSA is a direct regulator of salivary anti-IAV activity and is a determinant of individual differences. Additionally, after comparing the anti-IAV activity across the groups by age, anti-IAV activity in young people (aged 5-19 years) were lower than in adults aged 20-59 years and elderly people aged 60-79 years. Our study suggests that BSA levels in saliva may be important in preventing influenza infection.

Effect of Catechins on Upper Respiratory Tract Infections in Winter: A Randomized, Placebo-Controlled, Double-Blinded Trial.

Tea catechins are plant-derived compounds that improve immune functions. Previous randomized control trials have demonstrated the efficacy of primarily epi-type catechins against upper respiratory tract infections (URTIs). Green tea can be consumed in several ways, including popular bottled beverages. These beverages, however, require sterilization during manufacturing, which results in catechin isomerization. We conducted a randomized, double-blinded, placebo-controlled trial involving healthy Japanese participants to evaluate whether catechin consumption via bottled beverages has an alleviating effect on the duration and severity of URTIs in winter. The catechin group (490 mg catechin, 0.14%, containing 59% epi-type catechin, n = 55) showed reduced durations of running nose, nasal congestion, and headache, compared with the placebo group (0 mg catechin, n = 54; p = 0.013, 0.018, and <0.001, respectively). Furthermore, when considering physical symptoms, the duration of nasopharyngeal symptoms improved significantly in the catechin group (p < 0.001) compared with that in the control group. The daily consumption of catechin thus reduced the duration and severity of URTIs in healthy men and women. Humans are regularly exposed to several potential infectious threats, and the oral administration of heat-epimerized tea catechins might help prevent and reduce the severity of URTIs.

5,5-Dimethyl-3-(5-methyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-1-phenyl-3-(trifluoromethyl)-3,5,6,7-tetrahydro-1H-indole-2,4-dione, a potent inhibitor for mammalian elongase of long-chain fatty acids family 6: examination of its potential utility as a pharmacological tool.

Long-chain fatty acid elongases reside in the endoplasmic reticulum and are responsible for the rate-limiting step of the elongation of long-chain fatty acids. The elongase of long-chain fatty acids (ELOVL) family 6 (ELOVL6) is involved in the elongation of saturated and monosaturated fatty acids. Increased expression of ELOVL6 in ob/ob mice suggests a role for ELOVL6 in metabolic disorders. Furthermore, ELOVL6-deficient mice are protected from high-fat diet-induced insulin resistance, which suggests that ELOVL6 might be a new therapeutic target for diabetes. As reported previously, we developed a high-throughput screening system for fatty acid elongases and discovered lead chemicals that possess inhibitory activities against ELOVL6. In the present study, we examined in detail the biochemical and pharmacological properties of 5,5-dimethyl-3-(5-methyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-1-phenyl-3-(trifluoromethyl)-3,5,6,7-tetrahydro-1H-indole-2,4-dione (Compound-A), a potent inhibitor of ELOVL6. In in vitro assays, Compound-A dose-dependently inhibited mouse and human ELOVL6 and displayed more than 30-fold greater selectivity for ELOVL6 over the other ELOVL family members. In addition, Compound-A effectively reduced the elongation index of fatty acids of hepatocytes, suggesting that Compound-A penetrates the cell wall and inhibits ELOVL6. More importantly, upon oral administration to mice, Compound-A showed high plasma and liver exposure and potently reduced the elongation index of the fatty acids of the liver. This is the first study to report a potent and selective inhibitor of mammalian elongases. Furthermore, Compound-A seems to be a useful tool to further understand the physiological roles of ELOVL6 and to evaluate the therapeutic potential of an ELOVL6 inhibitor.

High-throughput assay for long chain fatty acyl-CoA elongase using homogeneous scintillation proximity format.

Elongase of very-long-chain fatty acid (Elovl) 6 is a rate-limiting enzyme that is responsible for the elongation of long-chain fatty acids such as palmitoic acid (C16). Elovl6 is abundantly expressed in liver and adipose tissue, and the expression levels in these tissues are up-regulated in obese animals. Furthermore, Elovl6-deficient mice display improved glucose homeostasis and insulin sensitivity, suggesting that Elovl6 might be a potential therapeutic target for metabolic disorders. From the drug discovery point of view, it is critical to establish a high-throughput screening (HTS) assay for the identification of therapeutic agents. Conventional assay methods for fatty acid elongases include an extraction step for respective radioactive products from the reaction mixtures, which is labor-intensive and not feasible for HTS. In this study, we utilized the acyl-coenzyme A (CoA) binding protein (ACBP) as a molecular probe to detect radioactive long-chain acyl-CoA, a direct product of Elovl6. Recombinant ACBP binds stearoyl-CoA but not malonyl-CoA, enabling specific detection of the radioactive product in the homogenous reaction mixture without the liquid extraction step. Finally, combination of ACBP and scintillation proximity assay beads led to specific detection of Elovl6 activity with appropriate window and reproducibility amenable to HTS (signal-to-background noise ratio of approximately 13.0-fold, Z' = 0.85). The assay system described here has the potential to enable identification of small compounds that modify fatty acid elongase activity and assessment of the therapeutic potential of acyl-CoA elongases.

Identification of positron emission tomography ligands for NPY Y5 receptors in the brain.

A series of trans-3-oxospiro[(aza)isobenzofuran-1(3H),1'-cyclohexane]-4'-carboxamide derivatives were synthesized and profiled for NPY Y5 binding affinity, brain and CSF penetrability in rats, and susceptibility to human and mouse P-glycoprotein transporters in order to develop a PET ligand. Compound 12b exhibited an acceptable profile for a PET ligand, and [(11)C]12b was successfully utilized in clinical settings as a Y5 PET ligand.

Identification of novel and orally active spiroindoline NPY Y5 receptor antagonists.

A series of spiroindoline-3,4'-piperidine derivatives were synthesized and evaluated for their binding affinities and antagonistic activities at Y5 receptors. Potent Y5 antagonists were tested for their oral bioavailabilities and brain penetration in rats. Some of the antagonists showed good oral bioavailability and/or good brain penetration. In particular, compound 6e was orally bioavailable and brain penetrant, and oral administration of 6e inhibited bPP-induced food intake in rats with a minimum effective dose of 10mg/kg.

Synthesis and evaluation of a novel 2-azabicyclo[2.2.2]octane class of long chain fatty acid elongase 6 (ELOVL6) inhibitors.

A series of novel 2-azabicyclo[2.2.2]octane derivatives was synthesized and evaluated as long chain fatty acid elongase 6 (ELOVL6) inhibitors. Screening of our corporate chemical collections against ELOVL6 resulted in the identification of lead 1. Exploratory chemistry efforts were applied to lead 1 to identify the orally available, potent, and selective ELOVL6 inhibitor 28a.

Discovery of trans-N-[1-(2-fluorophenyl)-3-pyrazolyl]-3-oxospiro[6-azaisobenzofuran-1(3H),1'-cyclohexane]-4'-carboxamide, a potent and orally active neuropeptide Y Y5 receptor antagonist.

A series of trans-3-oxospiro[(aza)isobenzofuran-1(3H),1'-cyclohexane]-4'-carboxamide derivatives were synthesized to identify potent NPY Y5 receptor antagonists. Of the compounds, 21j showed high Y5 binding affinity, metabolic stability and brain and cerebrospinal fluid (CSF) penetration, and low susceptibility to P-glycoprotein transporters. Oral administration of 21j significantly inhibited the Y5 agonist-induced food intake in rats with a minimum effective dose of 1mg/kg. This compound was selected for proof-of-concept studies in human clinical trials.

Identification of the Catechin Uptake Transporter Responsible for Intestinal Absorption of Epigallocatechin Gallate in Mice.

Many studies have shown that epigallocatechin gallate (EGCg) contribute to the health benefits of green tea, although its bioavailability is usually low. However, the mechanism underlying its intestinal absorption remains unclear. In human subjects, it has been reported that the bioavailability of EGCg increases after repeated oral catechin intake. We hypothesized that a certain uptake transporter was involved in this increase, and investigated a novel EGCg transporter. We first confirmed the increase in EGCg bioavailability in mice fed the catechin diet for two weeks. Then, in situ intestinal catechin infusion exhibited that the absorption of EGCg in the ileum was selectively increased in mice fed the catechin diet. A comprehensive analysis of plasma membrane proteins revealed 10 candidates for EGCg transporter, which were selectively increased in the ileum. EGCg uptake by a Xenopus laevis oocyte expressed with respective transporter revealed that oocytes microinjected with DTDST cRNA exhibited significantly higher EGCg uptake. Furthermore, uptake of EGCg by CHO-K1 cells stably expressing DTDST was significantly higher than that by mock cells, which was nullified by treating with a DTDST inhibitor. In conclusion, this study identified DTDST as a novel intestinal EGCg transporter that is upregulated after repeated oral catechin intake.

Green Tea Extracts Attenuate Brain Dysfunction in High-Fat-Diet-Fed SAMP8 Mice.

Unhealthy diet promotes progression of metabolic disorders and brain dysfunction with aging. Green tea extracts (GTEs) have various beneficial effects and alleviate metabolic disorders. GTEs have neuroprotective effects in rodent models, but their effects against brain dysfunction in models of aging fed unhealthy diets are still unclear. Here, we showed that GTEs attenuate high-fat (HF) diet-induced brain dysfunction in senescence-accelerated mouse prone-8 (SAMP8), a murine model of senescence. SAMP8 mice were fed a control diet, HF diet, or HF diet with 0.5% GTEs (HFGT) for four months. The HF diet reduced memory retention and induced amyloid ß1-42 accumulation, whereas GTEs attenuated these changes. In HF diet-fed mice, lipid oxidative stress, assessed by malondialdehyde levels, was increased. The levels of proteins that promote synaptic plasticity, such as brain-derived neurotrophic factor (BDNF) and postsynaptic density protein 95 (PSD95), were reduced. These alterations related to brain dysfunction were not observed in HFGT diet-fed mice. Overall, our data suggest that GTEs intake might attenuate brain dysfunction in HF diet-fed SAMP8 mice by protecting synaptic plasticity as well as via anti-oxidative effects. In conclusion, GTEs might ameliorate unhealthy diet-induced brain dysfunction that develops with aging.

Green tea extracts ameliorate high-fat diet-induced muscle atrophy in senescence-accelerated mouse prone-8 mice.

Muscle atrophy (loss of skeletal muscle mass) causes progressive deterioration of skeletal function. Recently, excessive intake of fats was suggested to induce insulin resistance, followed by muscle atrophy. Green tea extracts (GTEs), which contain polyphenols such as epigallocatechin gallate, have beneficial effects on obesity, hyperglycemia, and insulin resistance, but their effects against muscle atrophy are still unclear. Here, we found that GTEs prevented high-fat (HF) diet-induced muscle weight loss in senescence-accelerated mouse prone-8 (SAMP8), a murine model of senescence. SAMP8 mice were fed a control diet, an HF diet, or HF with 0.5% GTEs (HFGT) diet for 4 months. The HF diet induced muscle weight loss with aging (measured as quadriceps muscle weight), whereas GTEs prevented this loss. In HF diet-fed mice, blood glucose and plasma insulin concentrations increased in comparison with the control group, and these mice had insulin resistance as determined by homeostasis model assessment of insulin resistance (HOMA-IR). In these mice, serum concentrations of leukocyte cell-derived chemotaxin 2 (LECT2), which is known to induce insulin resistance in skeletal muscle, were elevated, and insulin signaling in muscle, as determined by the phosphorylation levels of Akt and p70 S6 kinases, tended to be decreased. In HFGT diet-fed mice, these signs of insulin resistance and elevation of serum LECT2 were not observed. Although our study did not directly show the effect of serum LECT2 on muscle weight, insulin resistance examined using HOMA-IR indicated an intervention effect of serum LECT2 on muscle weight, as revealed by partial correlation analysis. Accordingly, GTEs might have beneficial effects on age-related and HF diet-induced muscle weight loss, which correlates with insulin resistance and is accompanied by a change in serum LECT2.

Design and synthesis of the potent, orally available, brain-penetrable arylpyrazole class of neuropeptide Y5 receptor antagonists.

Novel arylpyrazole derivatives were synthesized and evaluated as neuropeptide Y (NPY) Y5 receptor antagonists. Compound (-)-7, which features a novel chiral 2,3-dihydro-1H-cyclopenta[a]naphthalene moiety, showed good binding affinity and antagonistic activity for the Y5 receptor. After intracerebroventricular administration in SD rats, (-)-7 significantly inhibited food intake that was induced by the centrally administered Y5-preferring agonist, bovine pancreatic polypeptide, but had only a negligible effect on NPY-induced feeding.

Discovery and characterization of a novel potent, selective and orally active inhibitor for mammalian ELOVL6.

The elongase of long chain fatty acids family 6 (ELOVL6) is a rate-limiting enzyme for the elongation of saturated and monounsaturated long chain fatty acids. ELOVL6 is abundantly expressed in lipogenic tissues such as liver, and its mRNA expression is up-regulated in obese model animals. ELOVL6 deficient mice are protected from high-fat-diet-induced insulin resistance, suggesting that ELOVL6 might be a new therapeutic target for diabetes. We previously identified an indoledione compound, Compound A, as the first inhibitor for mammalian ELOVL6. In this study, we discovered a novel compound, Compound B, and characterized its biochemical and pharmacological properties. Compound B has a more appropriate profile for use as a pharmacological tool compared to Compound A. Chronic treatment with Compound B in model animals, diet-induced obesity (DIO) and KKAy mice, showed significant reduction in hepatic fatty acid composition, suggesting that it effectively inhibits ELOVL6 activity in the liver. However, no improvement in insulin resistance by ELOVL6 inhibition was found in these model animals. Further studies need to address the impact of ELOVL6 inhibition on pharmacological abnormalities in several model animals. This is the first report on pharmacology data from chronic studies using a selective ELOVL6 inhibitor. Compound B appears to be a useful tool to further understand the physiological roles of ELOVL6 and to evaluate the therapeutic potential of ELOVL6 inhibitors.

Development of a high-density assay for long-chain fatty acyl-CoA elongases.

We established a convenient assay method for measuring elongation of very long chain fatty acids (ELOVLs) using a Unifilter-96 GF/C plate. The Unifilter GF/C plate preferentially interacts with hydrophobic end products of ELOVLs (i.e., long chain fatty acid), with minimal malonyl-CoA (C2 unit donor for fatty acid elongation) interaction. This new method results in the quick separation and detection of [(14)C] incorporated end products (e.g., [(14)C] palmitoyl-CoA) from reaction mixtures containing excessive amounts of [(14)C] malonyl-CoA. In the Unifilter-96 GF/C plate assay, recombinantly expressed human ELOVLs (i.e., ELOVL1,-2,-3,-5 and -6) displayed appreciable assay windows (>2-fold vs. mock-transfected control), enabling us to conduct comprehensive substrate profiling of ELOVLs. The substrate concentration profile of ELOVL6 in the Unifilter-96 GF/C plate assay is consistent with that obtained from the conventional liquid extraction method, thus, supporting the reliability of the Unifilter-96 GF/C plate assay. We then examined the substrate specificities of ELOVLs in a comprehensive fashion. As previously reported, ELOVL1, -3 and -6 preferably elongated the saturated fatty acyl-CoAs while ELOVL2 and ELOVL5 preferentially elongated the polyunsaturated fatty acyl-CoAs. This further confirms the Unifilter-96 GF/C plate assay reliability. Taken together, our newly developed assay provides a convenient and comprehensive assay platform for ELOVLs, allowing investigators to conduct high density screening and characterization of ELOVLs chemical tools.

(-)-Ternatin inhibits adipogenesis and lipid metabolism in 3T3-L1 cells.

(-)-Ternatin, a highly N-methylated cyclic peptide, inhibits fat accumulation in 3T3-L1 cells and reduces fat mass in mice. However, the mechanism for its anti-adipogenic effect has remained unknown. To examine the mechanism used by (-)-ternatin to inhibit adipocyte differentiation, we examined the effects of (-)-ternatin and [l-Ala(4)]ternatin, an inactive analog of (-)-ternatin, on the expression of adipocyte markers and lipogenic enzymes. We found that (-)-ternatin potently reduced mRNA expression of several adipocyte markers in a dose-dependent manner, whereas [l-Ala(4)]ternatin showed no effects. At the immediate early phase, (-)-ternatin, but not [l-Ala(4)]ternatin, reduced the expression of Srebp1c, Fas, Acc2 and C/EBP-alpha while showing no effects on C/EBP-beta and C/EBP-delta. These results suggest that (-)-ternatin affects the mid-to late differentiation stages of adipocytes. Consistent with the decreased expression of lipogenic enzymes, (-)-ternatin potently inhibited triglyceride synthesis. Intriguingly, (-)-ternatin also inhibited triglyceride synthesis in rat primary hepatocytes, suggesting that the potential action sites for (-)-ternatin are shared by adipocytes and liver. Although the target molecule of (-)-ternatin remains unknown, our data suggest that (-)-ternatin and its potential target might provide a new therapeutic approach to metabolic disorders.

Synthesis and biological evaluation of a novel 3-sulfonyl-8-azabicyclo[3.2.1]octane class of long chain fatty acid elongase 6 (ELOVL6) inhibitors.

Long chain fatty acid elongase 6 (ELOVL6) catalyzes the elongation of long chain fatty acyl-CoAs and is a potential target for the treatment of metabolic disorders. The ultrahigh throughput screen of our corporate chemical collections resulted in the identification of a novel 3-sulfonyl-8-azabicyclo[3.2.1]octane class of ELOVL6 inhibitor 1a. Optimization of lead 1a led to the identification of the potent, selective, and orally available ELOVL6 inhibitor 1w.

Synthesis and evaluation of a novel indoledione class of long chain fatty acid elongase 6 (ELOVL6) inhibitors.

Novel indoledione derivatives were synthesized and evaluated as long chain fatty acid elongase 6 (ELOVL6) inhibitors. Systematic optimization of an indole class of lead 1 led to the identification of potent ELOVL6 selective inhibitors. Representative inhibitor 37 showed sustained plasma exposure and good liver penetrability in mice. After oral administration, 37 potently inhibited ELOVL6 activity in the liver in mice.