Mitsuaki Sano

Formation of antioxidants from (-)-epigallocatechin gallate in mild alkaline fluids, such as authentic intestinal juice and mouse plasma.

The oxidative dimerization of (-)-epigallocatechin gallate (EGCG), the major catechin of tea leaves (Camellia sinensis L.), in authentic intestinal juice (pH 8.5) and mouse plasma (pH 7.8) was investigated. EGCG was unstable in the alkaline solutions over pH 7.4. The content of EGCG was decreased to 19.4% and 60.7% at 5 minutes in the intestinal juice and plasma, respectively. Three products-P-1 (theasinensin A), P-2 (a new dimerized product reported in a previous paper), and P-3 (theasinensin D, a rotational isomer of P-1)-were detected in these fluids. The sum of the molar contents of the three products formed after 5 minutes of incubation at 37 degrees C corresponded to 35.1% and 21.9% of the degraded molar content of EGCG, respectively. These dimerization products of EGCG would be formed by the dehydrogenation and decarboxylation of EGCG under oxidative conditions in alkaline solutions. The formation of P-2 was greater than that of P-1 and of P-3 at 30 minutes of incubation in the intestinal juice and mouse plasma. Fe(2+)-chelating activities of the three products were much higher than that of EGCG, and superoxide anion radical-scavenging activity of P-2 was also significantly higher than that of EGCG. The absorbance of P-2 administered to male ddY mice was studied. The content of P-2 in mouse plasma was less than that of administration of EGCG, but P-2 was absorbed quickly within 30 minutes and metabolized slowly. These dimerization products of EGCG are expected to contribute to in vivo antioxidative activities enhanced by tea drinking.

O-methylated catechins from tea leaves inhibit multiple protein kinases in mast cells.

Tea contains a variety of bioactive compounds. In this study, we show that two O-methylated catechins, (-)-epigallocatechin-3-O-(3-O-methyl) gallate and (-)-epigallocatechin-3-O-(4-O-methyl) gallate, inhibit in vivo mast cell-dependent allergic reactions more potently than their nonmethylated form, (-)-epigallocatechin-3-O-gallate. Consistent with this, these O-methylated catechins inhibit IgE/Ag-induced activation of mouse mast cells: histamine release, leukotriene release, and cytokine production and secretion were all inhibited. As a molecular basis for the catechin-mediated inhibition of mast cell activation, Lyn, Syk, and Bruton’s tyrosine kinase, the protein tyrosine kinases, known to be critical for early activation events, are shown to be inhibited by the O-methylated catechins. In vitro kinase assays using purified proteins show that the O-methylated catechins can directly inhibit the above protein tyrosine kinases. These catechins inhibit IgE/Ag-induced calcium response as well as the activation of downstream serine/threonine kinases such as Akt and c-Jun N-terminal kinase. These observations for the first time have revealed the molecular mechanisms of antiallergic effects of tea-derived catechins.

Simultaneous determination of twelve tea catechins by high-performance liquid chromatography with electrochemical detection

A high-performance liquid chromatographic method with electrochemical detection was developed for the determination of twelve tea catechins including four major catechins: epicatechin (EC), epigallocatechin (EGC), epicatechin gallate (ECG) and epigallocatechin gallate (EGCG); four of their epimers at the C-2 position, C, GC, CG and GCG; and four methylated catechin derivatives, epigallocatechin-3-O-(3-O-methyl)gallate, gallocatechin-3-O-(3-O-methyl)gallate, epigallocatechin-3-O-(4-O-methyl)gallate and epicatechin-3-O-(3-O-methyl)gallate. These catechins were separated on an ODS C18 reversed-phase column by isocratic elution with 0.1 M NaH2PO4 buffer (pH 2.5)-acetonitrile (87:13) containing 0.1 mM EDTA.2Na. The detection limits (S/N = 3) of these catechins were approximately 10-40 pmol ml-1 at an applied voltage of 600 mV. Extracting these catechins from tea leaf powder with H2O-acetonitrile (1:1) at 30 degrees C for 40 min inhibited the epimerization at C-2 significantly from these epicatechins compared to extraction with hot water at 90 degrees C. This analytical method is sensitive to and appropriate for the simultaneous determination of various biologically active catechins in green tea.

Effects of long-term dietary supplement of tea polyphenols on lipid peroxide levels in rats

We examined the effects of tea polyphenols (TP) on the body weight gain and contents of lipids and lipid peroxides in rat plasma, kidney and liver. The supplementation of TP (0.5% and 1.0%) in the diet was performed from weanling (3 weeks of age) to 19 months old. A significant suppression of body weight gain was observed only at 7–11 weeks of age, but there were no significant changes in liver and kidney weight between control group and TP-feeding group (TP group). Daily food intake of animals in two TP groups was almost the same as the control group.In the older rats (13–19 months of age), thiobarbituric acid reactive substances (TBARS) in the plasma in 1.0% TP group were significantly lower than that of the control g roup (p<0.01). The contents of plasma lipids (triglyceride, total cholesterol, and phospholipid) in 1.0% TP group at 19 months old were significantly lower as compared with the control group (p<0.05). There was a positive correlation in an age group 13–19 months old between these lipid contents and TBARS.These results suggest that long-term dietary supplementation of TP caused the decrease of plasma TBARS, mainly due to the hypolipidemic activity and antioxidative effect of TP in vivo.

Antioxidants from Lespedeza homoloba. (I)

The stems of Lespedeza homoloba yielded eight new and three known phenolic compounds. Their structures have been elucidated on the basis of their spectral data. These compounds had strong antioxidative activity against lipid peroxidation in the rat brain homogenate test. 3,9-Dihydroxypterocarp-6a-en and lespedezol A2 showed significant antiallergic activity in allergic (type I) mice.

Different onsets of oxidative damage to DNA and lipids in bone marrow and liver in rats given total body irradiation.

We examined time-dependent changes in antioxidant vitamins and oxidative damage to DNA and lipids in the bone marrow, liver, and plasma of rats given total body irradiation (TBI) with X-rays at 3 Gy. The oxidative damage to DNA and lipids was evaluated by measuring increases of 8-hydroxydeoxyguanosine (8OHdG) in DNA and 4-hydroxy-2-nonenal (HNE), respectively. After the TBI, marked increases in 8OHdG and HNE were detected at 3 to 5 h in the bone marrow, while gradual increases in these parameters were detected after a few days in the liver. These changes in 8OHdG and HNE were well correlated within each tissue. In the bone marrow, levels of both vitamin C and vitamin E were decreased by the TBI; however, the changes in vitamin C were earlier and greater than those in vitamin E. In the liver, the level of vitamin C did not decrease, but that of vitamin E decreased due to the TBI. Changes in HNE, vitamin C, and vitamin E in the plasma were similar to those in the liver. Within each tissue, the time of decrease in antioxidants was almost the same as that of the increase in oxidative damage. An increase in total iron due to the TBI was also detected in these tissues. In particular, the total iron in the bone marrow was markedly increased at a few hours after the TBI, with a slight increase in transferrin and no increase in ferritin. Exposure studies performed on cells or isolated DNA showed that an increase in 8OHdG was detected immediately after irradiation at more than 100 Gy in bone marrow cells and at less than 10 Gy in isolated DNA, suggesting that an increase in 8OHdG is undetectable even in bone marrow immediately after the TBI at 3 Gy. These results indicate that the onset of oxidative damage to DNA and lipids was delayed after TBI at 3 Gy, that it was quite different in the bone marrow and the liver, and that an increase in iron and decrease in antioxidant vitamins were involved in the mechanism of oxidative damage.

Fluctuation of lipid peroxides and related enzyme activities at time of stroke in stroke-prone spontaneously hypertensive rats.

The levels of lipid peroxides, determined as thiobarbituric acid reactive substances (TBARS), and the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), were examined in theblood from stroke-prone spontaneously hypertensive rats (SHRSP), with and without cerebral lesions, and normotensive Wistar Kyoto (WK)rats. The levels of TBARS in the blood from healthy SHRSP were not significantly different from those of WK rats, while the values of SHRSP (male) with stroke were more than twice as high as those of healthy SHRSP. The activities of SOD and GSH-Px in stroke SHRSP were also statistically different from those of healthy SHRSP.

Fluorescence detection of glutathione S conjugate with aldehyde by high-performance liquid chromatography with post-column derivatization

A high-performance liquid chromatographic method with post-column derivatization was developed for the detection of giutathione (GSH) S conjugates with aliphatic aldehydes produced during lipid peroxidation. The GSH S conjugates were separated on a reversed-phase C18 column coated with synthetic polymer, using a mobile phase consisting of a methanol–phosphate buffer gradient system, and were then treated with o-phthalaldehyde (OPA) in the reaction coil. The detection of the OPA derivatives was effected by fluorimetry (λex= 340 nm; λem= 450 nm). The detection limits calculated for some aldehydes added to the reaction system were approximately 12–111 pmol (signal-to-noise ratio = 3). This is a simple and sensitive method for the detection of GSH S conjugates with aldehydes in biological samples. By using this method, it was found that the conjugates were readily produced in rat red blood cells incubated with some alkenals and hydroxyalkenals at 37 °C for 60 min.

Potentiation of oxidative damage to rat red blood cells by the concurrent presence of t-butyl hydroperoxide and bromotrichloromethane.

Recently potentiation of oxidative damage in rat red blood cells (rRBC) incubated with t-butylhydroperoxide (BHP) in combination with bromotrichloromethane (BrCCI3) was demonstrated. The mechanism by which this combination (BrCCI3/BHP) potentiates the oxidative damage to rRBC was investigated in this study. When rRBC were incubated with 0.1 mM BHP, 0.5 mM BrCCI3, or the two combined, BrCCI3/BHP-potentiated lipid peroxidation and hemolysis were further enhanced under anaerobic conditions. However, the potentiation of lipid peroxidation was abolished by heating or trypsin digestion of rRBC. Electron spin resonance (ESR) studies demonstrated an increase of alkoyl radical induced by BrCCI3/BHP in rRBC, and this increase was abolished by heating or predigestion of hemolysates with trypsin. The inhibition of lipid peroxidation by diphenylamine (which reacts with alkoxyl radicals but not peroxyl radicals) suggests an important role of alkoxyl radicals. Overall, the present findings demonstrate that the increase in radical-related oxidative damage, possibly mediated by proteinlike materials, may be at least partially responsible for the potentiation of damage to rRBC induced by BrCCI3/BHP, and perhaps by BrCCI3. Although the in vivo significance of these results remains to be investigated, it seems likely that halocarbon toxicity may be amplified by elevated levels of lipid peroxide in blood.