The effect of orally administrated gamma-aminobutyric acid (GABA) on relaxation and immunity during stress has been investigated in humans. Two studies were conducted. The first evaluated the effect of GABA intake by 13 subjects on their brain waves. Electroencephalograms (EEG) were obtained after 3 tests on each volunteer as follows: intake only water, GABA, or L-theanine. After 60 minutes of administration, GABA significantly increases alpha waves and decreases beta waves compared to water or L-theanine. These findings denote that GABA not only induces relaxation but also reduces anxiety. The second study was conducted to see the role of relaxant and anxiolytic effects of GABA intake on immunity in stressed volunteers. Eight acrophobic subjects were divided into 2 groups (placebo and GABA). All subjects were crossing a suspended bridge as a stressful stimulus. Immunoglobulin A (IgA) levels in their saliva were monitored during bridge crossing. Placebo group showed marked decrease of their IgA levels, while GABA group showed significantly higher levels. In conclusion, GABA could work effectively as a natural relaxant and its effects could be seen within 1 hour of its administration to induce relaxation and diminish anxiety. Moreover, GABA administration could enhance immunity under stress conditions.

An extract of Japanese green tea, one of the most popular drinks in Japan, was an inhibitor of the growth of Streptococcus mutans, a bacterium responsible for causing dental caries. The analysis of the extract revealed that the main antibacterial componentsof the extract were several polyphenolic compounds, especially gallocatechin (GC), epigallocatechin (EGC), and epigallocatechin gallate (EGCg). GC was the most active component and its minimum inhibitory concentration against the bacterium was around 250 ug per ml.

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The structure and antimicrobial function of hen egg white lysozyme was investigated by means of thermal denaturation at 80 °C (pH 7.2), which leads to irreversible denaturation. With an increase in the heating time (up to 30 min) of lysozyme, the soluble fraction showed progressive decrease in its enzyme activity that coincided with the formation of a slower migrating band on the acid PAGE. Fluorescence spectra revealed that, as the extent of denaturation increases, the surface hydrophobicity and the exposure of tryptophan residues were greatly promoted. In parallel to these conformational changes of lysozyme there has been consistent increase in its antimicrobial activities against Gram-negative bacteria, with no detrimental effect on its inherent action to Gram-positive bacteria. Interestingly, lysozyme heated for 20 min, devoid of enzyme activity (HDLz), killed Escherichia coli K12 in a dose-dependent manner, while its bactericidal activity to Staphylococcus aureus was almost similar to that of the native lysozyme. The binding capacity of HDLz to membrane fractions of E. coli K12 was greatly promoted, particularly to the inner membrane, as determined by ELISA. The HDLz permeabilized liposomal membranes made from E. coli phospholipids, as demonstrated by calcein efflux, in a protein concentration-dependent manner. Good correlations between the degree of heat inactivation of lysozyme (or dimerization), increased hydrophobicity, and enhanced bactericidal activity against Gram-negative E. coli K12 were observed. The results of this study, first of all, suggest that susceptibility of Gram-negative or even Gram-positive bacteria to lysozyme is independent of enzymatic activity. It is likely that denatured lysozyme, e.g., the dimeric form, has an intrinsic structural motif which is generally lethal to the bacteria through membrane perturbation. Keywords: Lysozyme; denaturation; conformational changes; antimicrobial action; membrane interaction; liposome

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The effect of green tea catechin supplementation on antioxidant capacity of human plasma was investigated. Eighteen healthy male volunteers who orally ingested green tea extract (254 mg of total catechins/subject) showed 267 pmol of epigallocatechin-3-gallate (EGCg) per milliliter of plasma at 60 min after administration. The plasma phosphatidylcholine hydroperoxide (PCOOH) levels attenuated from 73.7 pmol/mL in the control to 44.6 pmol/mL in catechin-treated subjects, being correlated inversely with the increase in plasma EGCg level. The results suggested that drinking green tea contributes to prevent cardiovascular disease by increasing plasma antioxidant capacity in humans.

Previous investigations have demonstrated that green tea polyphenols and partially hydrolyzed guar gum as dietary fiber have antioxidative and hypolipidemic activity, respectively, supporting their reduction of risk factors in the course of diabetic nephropathy via a hypoglycemic effect and ameliorating the decline of renal function through their combined administration to rats with subtotal nephrectomy plus streptozotocin (STZ) injection. As a further study, we examined whether (Ϫ)-epigallocatechin 3-O-gallate (EGCg), the main polyphenolic compound, could ameliorate the development of diabetic nephropathy. Rats with subtotal nephrectomy plus STZ injection were orally administrated EGCg at doses of 25, 50, and 100 mg/kg body weight/day. After a 50-day administration period, EGCgtreated groups showed suppressed hyperglycemia, proteinuria, and lipid peroxidation, although there were only weak effects on the levels of serum creatinine and glycosylated protein. Furthermore, EGCg reduced renal advanced glycation endproduct accumulation and its related protein expression in the kidney cortex as well as associated pathological conditions. These results suggest that EGCg ameliorates glucose toxicity and renal injury, thus alleviating renal damage caused by abnormal glucose metabolism-associated oxidative stress involved in renal lesions of diabetic nephropathy.

The antimicrobial mechanism and structural changes of hen egg white lysozyme irreversibly inactivated at 80 °C and at different pHs were investigated. We found that heat denaturation of lysozyme at increasing temperatures for 20 min at pH 6.0 results in progressive loss of enzyme activity while greatly promotes its antimicrobial action to Gram-negative bacteria. Interestingly, lysozyme devoid of enzyme activity (heated at 80 °C and pH 7.0 or at pH 6.0 over 90 °C) exhibited strong bactericidal activity against Gram-negative and -positive bacteria, suggesting action independent of catalytic function. The most potent antimicrobial lysozyme to either Gram-negative or -positive bacteria was that heated at 80 °C and pH 6.0 (HLz80/6), retaining 50% of the native enzymatic activity, which exhibited a 14-fold increase in surface hydrophobicity, with two exposed thiol groups. HLz80/6-induced agglutination coincided with severe reduction in colony-forming ability of the susceptible bacteria in a dose-dependent manner. Denatured lysozyme HLz80/6 showed promoted binding capacity to peptidoglycan of Staphylococcus aureus and lipopolysaccharide of Escherichia coli as assessed by ELISA. Addition of HLz80/6 to E. coli phospholipid vesicles resulted in a blue shift in the intrinsic tryptophan fluorescence accompanied by an increase in the size of the vesicles, indicating enhanced protein−membrane binding and subsequent fusion of liposomes. Direct membrane damage of E. coli membrane by HLz80/6 was revealed by electron microscopy observation. Thus, the results introduce an interesting finding that partial unfolding of lysozyme with the proper acquisition of the hydrophobic pocket to the surface can switch its antimicrobial activity to include Gram-negative bacteria without a detrimental effect on the inherent bactericidal effect against Gram-positive ones. The data suggest that the unique antimicrobial action of unfolded lysozyme attributes to membrane binding and subsequent perturbation of its functions. Keywords: Lysozyme; conformational changes; antimicrobial action; agglutination; membrane interaction and fusion