The polyphenol (-)-epicatechin gallate disrupts the secretion of virulence-related proteins by Staphylococcus aureus

Aim: (?)‐Epicatechin gallate (ECg) modifies the morphology, cell wall architecture and β‐lactam antibiotic susceptibility of Staphylococcus aureus. As these effects result primarily from intercalation into the bacterial cytoplasmic membrane, the capacity of ECg to modulate the secretion of two key staphylococcal virulence factors, coagulase and α‐toxin, was examined. Methods and Results: Bioassays were used to determine coagulase and haemolysin activity in culture supernatants of a number of S. aureus isolates grown in the presence and absence of ECg; α‐toxin secretion was also evaluated by immunoblotting. Growth in ECg reduced the levels of activity of both proteins in culture supernatants; the effects could only be partly explained by ECg‐mediated inhibition of bioactivity and by induction of secreted proteases. Conclusion: ECg suppresses the secretion of coagulase and α‐toxin by clinical isolates of S. aureus. Significance and Impact of the Study: The observation that secretion of key components of staphylococcal virulence can be compromised by a naturally occurring polyphenol supports the notion that ECg and related compounds may have therapeutic utility for the control of infections that are currently difficult to treat due to the propensity of methicillin‐resistant S. aureus to accumulate antibiotic resistance genes.     

Suppressive effect of nobiletin and epicatechin gallate on fructose uptake in human intestinal epithelial Caco-2 cells

Abstract

Inhibition of excessive fructose intake in the small intestine could alleviate fructose-induced diseases such as hypertension and non-alcoholic fatty liver disease. We examined the effect of phytochemicals on fructose uptake using human intestinal epithelial-like Caco-2 cells which express the fructose transporter, GLUT5. Among 35 phytochemicals tested, five, including nobiletin and epicatechin gallate (ECg), markedly inhibited fructose uptake. Nobiletin and ECg also inhibited the uptake of glucose but not of L-leucine or Gly-Sar, suggesting an inhibitory effect specific to monosaccharide transporters. Kinetic analysis further suggested that this reduction in fructose uptake was associated with a decrease in the apparent number of cell-surface GLUT5 molecules, and not with a change in the affinity of GLUT5 for fructose. Lastly, nobiletin and ECg suppressed the permeation of fructose across Caco-2 cell monolayers. These findings suggest that nobiletin and ECg are good candidates for preventing diseases caused by excessive fructose intake.     

Carbocations in the synthesis of prostaglandins by the cyclooxygenase of pgh synthase? a radical departure!

Evidence already available is used to demonstrate that although prostaglandin G/H synthase hydroxylates arachidonic acid through radical intermediates, it effects cyclizations through a carbocation center at C-10. This is produced following migration of H to the initial radical at C-13 and a 1epsilon oxidation. Under orbital symmetry control, the cyclizations can give only the ring size and trans stereochemistry actually observed. After cyclization, the H-shift reverses to take the sequence back into current radical theory for hydroxylation at C-15. Thus 10,10-difluoroarachidonic acid cannot be cyclized, although it can be hydroxylated. Acetylation of Ser516 in the isoform synthase-2 is considered to oppose carbocation formation and/or H-migration and so prevent cyclizations while permitting hydroxylations; the associated inversion of chirality at C-15 can then readily be accommodated without the change in conformation required by other schemes. Suicide inhibition occurs when carbocations form stable bonds upon (thermal) contact with adjacent heteroatoms, etc. Because the cyclooxygenase and peroxidase functions operate simultaneously through the same heme, phenol acts as reducing cosubstrate for the cyclooxygenase, thus enabling it to promote PGG2 production and protect the enzyme from oxidative destruction.     

分光光度法测定金荞麦(-)-表儿茶素含量的方法研究

为建立香荚兰素-盐酸分光光度法对表儿茶素含量的测定方法,实验分别以乙醇和水作溶剂,在34.79～208.74μg范围内取样置50 mL容量瓶测定,结果发现:①以乙醇作溶剂时,测定波长为508 nm,1%香荚兰浓盐酸添加量应超过40mL(CV≤1.86%),测定时间以40 min后为宜(CV≤2.65%),标准曲线线性关系(R=0.999 3)与精确度(CV=2.66%)、准确度(P0.05)良好。②以水作溶剂时,测定波长为504 nm,显色反应不稳定,标准曲线线性关系(R=0.988 1)也较一般。③采用乙醇作溶剂时,测得金荞麦块根中(-)-表儿茶素类物质含量占干物质的2.22%(CV≤3.90%),该法操作简便,灵敏度高,重现性好。     

Mechanisms of the antinociceptive action of (?) Epicatechin obtained from the hydroalcoholic fraction of Combretum leprosum Mart & Eic in rodents

ABSTRACT: BACKGROUND: The mechanisms of the antinociceptive activity of () epicatechin (EPI), a compound isolated from the hydroalcoholic fraction of Combreum leprosum Mart & Eicher. METHODS: were assessed in the model of chemical nociception induced by glutamate (20 mumol/paw). To evaluate the mechanisms involved, the animals , male Swiss mice (25-30 g), received EPI (50 mg/kg p.o.) after pretreatment with naloxone (2 mg/kg s.c. opioid antagonist), glibenclamide (2 mg/kg s.c. antagonist K + channels sensitive to ATP), ketanserin (0.3 mg/kg s.c. antagonist of receptor 5-HT2A), yoimbine (0.15 mg/kg s.c. alpha2 adrenergic receptor antagonist), pindolol (1 mg/kg s.c. 5-HT1a/1b receptor antagonist), atropine (0.1 mg/kg s.c. muscarinic antagonist) and caffeine (3 mg/kg s.c. adenosine receptor antagonist), ondansetron (0.5 mg/kg s.c. for 5-HT3 receptor) and L-arginine (600 mg/kg i.p.). RESULTS: The antinociceptive effect of EPI was reversed by pretreatment with naloxone and glibenclamide, ketanserin, yoimbine, atropine and pindolol, which demonstrates the involvement of opioid receptors and potassium channels sensitive to ATP, the serotoninergic (receptor 5HT1A and 5HT2A), adrenergic (receptor alpha 2) and cholinergic (muscarinic receptor) systems in the activities that were observed. The effects of EPI, however, were not reversed by pretreatment with caffeine, L-arginine or ondansetron, which shows that there is no involvement of 5HT3 receptors or the purinergic and nitrergic systems in the antinociceptive effect of EPI. In the Open Field and Rotarod test, EPI had no significant effect, which shows that there was no central nervous system depressant or muscle relaxant effect on the results. CONCLUSIONS: This study demonstrates that the antinociceptive activity of EPI in the glutamate model involves the participation of the opioid system, serotonin, adrenergic and cholinergic.     

Effect of new antioxidant cysteinyl-flavanol conjugates on skin cancer cells

Novel catechin derivatives obtained from grape procyanidins and l-cysteine scavenge free radicals by hydrogen atom donation, rather than electron transfer, and reduce cell viability in A375 and M21 melanoma cells. In particular, 4beta-(S-cysteinyl)epicatechin 3-O-gallate has a free radical scavenging capacity as strong as that of tea (-)-epigallocatechin gallate and causes a significant S-phase cell-cycle arrest in both cell lines at doses higher than 100 microM. The other cysteinyl compounds do not affect normal cell cycle distribution. The gallate derivative also induces apoptosis in melanoma cells more strongly than the other derivatives and the parent (-)-epicatechin do. The gallate compound seems to trigger nuclear condensation and fragmentation, which is confirmed by DNA laddering. Interestingly, they do not induce apoptosis in keratinocytes (HaCaT).     

Erythrocyte sodium/hydrogen exchange inhibition by (-) epicatechin

Epicatechin, a flavonoid belonging to the group of compounds collectively called catechins, have been reported to possess insulin-like properties. Besides their anti-diabetic properties, catechins also show growth inhibition. Since cytosolic pH (pHi) plays a role in cell proliferation and the Na/H exchanger (NHE) is the major pH (pHi) regulatory mechanism, we undertook in vitro studies with human erythrocytes to examine the effect of (-) epicatechin (EC) on the NHE1 isoform. NHE activity was measured in eight healthy volunteers, eight type 1 diabetics, and nine type 2 diabetics, following 30 min incubations at 37 degrees C with either 1 mM epicatechin, 10(-9) M insulin or solvent alone. NHE activity was elevated in both groups of patients (P< 0.05). Epicatechin caused a 93% decrease in Na/H antiport activity in health controls, 89 and 86% in type 1 and type 2 diabetics, respectively (P< 0.001). Insulin caused a 36% decrease in antiport activity only in the type 2 diabetic group (P< 0.05). The strong inhibition of erythrocyte NHE1 (the ubiquitously present isoform) by epicatechin may have important implications. NHE1 inhibition could be one of the major mechanisms underlying the antiproliferative effects of catechins.     

乙醇提取金荞麦(-)表儿茶素类活性物质的工艺

通过单因素试验法,经F检验和新复极差法(SSR法)比较,先后确定了乙醇浓度、提取温度、料液比、提取时间的大致范围;再通过四因素三水平的正交试验,得到优化后的提取工艺为乙醇体积分数60%、提取温度20℃、料液比(g/mL)1∶18、提取时间30 min,得到金荞麦(-)表儿茶素类活性物质的平均提取率达2.5%。工艺简单合理,可应用于金荞麦(-)表儿茶素单体产业化的前期生产。     

A novel approach to enhancing cellular glutathione levels

GSH and GSH-associated metabolism provide the major line of defense for the protection of cells from oxidative and other forms of toxic stress. Of the three amino acids that comprise GSH, cysteine is limiting for GSH synthesis. As extracellularly cysteine is readily oxidized to form cystine, cystine transport mechanisms are essential to provide cells with cysteine. Cystine uptake is mediated by system x(c)(-), a Na(+)-independent cystine/glutamate antiporter. Inhibition of system x(c)(-) by millimolar concentrations of glutamate, a pathway termed oxidative glutamate toxicity, results in GSH depletion and nerve cell death. Recently, we described a series of compounds derived from the conjugation of epicatechin (EC) with cysteine and cysteine derivatives that protected nerve cells in culture from oxidative glutamate toxicity by maintaining GSH levels. In this study, we characterize an additional EC conjugate, cysteamine-EC, that is 5- to 10-fold more potent than the earlier conjugates. In addition, we show that these EC conjugates maintain GSH levels by enhancing the uptake of cystine into cells through induction of a disulfide exchange reaction, thereby uncoupling the uptake from system x(c)(-). Thus, these novel EC conjugates have the potential to enhance GSH synthesis under a wide variety of forms of toxic stress.