Cardioprotective effect of edaravone against ischaemia-reperfusion injury in the rabbit heart before, during and after reperfusion treatment.

The free radical scavenger edaravone is able to stimulate prostacyclin release and inhibit the lipoxygenase pathway in the arachidonic acid cascade. The effect of edaravone administration on myocardial damage in rabbit hearts subjected to ischaemia-reperfusion was examined at different times relative to reperfusion. All rabbits underwent sustained coronary artery occlusion for 30 min followed by 3 h of reperfusion. Rabbits were divided into the following groups: control; early (3 mg/kg edaravone IV 10 min before reperfusion); immediate (3 mg/kg edaravone IV immediately after the start of reperfusion); and late (3, 6 or 10 mg/kg edaravone IV 5 min after the start of reperfusion). Single bolus administration of edaravone 10 min before reperfusion or immediately upon initiation of reperfusion appears to be associated with reductions in infarction size and the percentage of apoptotic cells, but treatment with edaravone 5 min after initiation of reperfusion does not appear to have this protective effect.

Effect of vitamin C on common cold: randomized controlled trial.

OBJECTIVE: To investigate the relationship between the common cold and vitamin C supplementation. DESIGN: A double-blind, 5-year randomized controlled trial. SETTING: A village in Akita prefecture, one of the regions in Japan with the highest mortality from gastric cancer. SUBJECTS: Participants in annual screening programs for circulatory diseases conducted under the National Health and Welfare Services Law for the Aged, and diagnosed as having atrophic gastritis. Of the 439 eligible subjects, 144 and 161 were assigned to receive 50 or 500 mg of vitamin C, respectively, after protocol amendment. During the supplementation phase, 61 dropped out, and 244 completed the trial. INTERVENTION: Daily vitamin C supplementation of 50 mg (low-dose group) or 500 mg (high-dose group). RESULTS: Total number of common colds (per 1000 person-months) was 21.3 and 17.1 for the low- and high-dose groups, respectively. After adjustment for several factors, the relative risks (95% confidence interval (CI)) of suffering from a common cold three or more times during the survey period was 0.34 (0.12-0.97) for the high-dose group. No apparent reduction was seen for the severity and duration of the common cold. CONCLUSION: A randomized, controlled 5-year trial suggests that vitamin C supplementation significantly reduces the frequency of the common cold but had no apparent effect on the duration or severity of the common cold. However, considering several limitations due to protocol amendment, the findings should be interpreted with caution.

Additive antitumour effect of the epidermal growth factor receptor tyrosine kinase inhibitor gefitinib (Iressa, ZD1839) and the antioestrogen fulvestrant (Faslodex, ICI 182,780) in breast cancer cells.

A high expression level of epidermal growth factor receptor (EGFR)/HER1 has been suggested to lead to a shorter survival time and resistance to endocrine therapy in patients with breast cancer. To test the hypothesis that inhibition of the EGFR signalling pathway affects the antitumour effect of endocrine therapy, an EGFR tyrosine kinase inhibitor (EGFR-TKI), gefitinib, and an oestrogen receptor (ER) antagonist, fulvestrant, were administered to human breast cancer cells. A total of five human breast cancer cell lines were used. The effects of single or combined treatments with gefitinib and/or fulvestrant on cell growth, cell cycle progression and apoptosis were analysed. Changes in the expression levels of cyclin-dependent kinase inhibitors, p21 and p27, an antiapoptotic factor, Bcl-2, and a proapoptotic factor, Bax, were also investigated. All cell lines tested were sensitive to gefitinib (50% growth inhibitory concentration, 10-28.5 microM). Breast cancer cell lines with a high expression level of HER1 or HER2 were more sensitive to gefitinib than the others. Gefitinib induced a significant G1-S blockade in ER-positive KPL-3C cells. Gefitinib induced significant apoptosis in HER1-overexpressing MDA-MB-231 cells. Gefitinib additively increased the antitumour effect of fulvestrant in all three ER-positive cell lines in a medium supplemented with 17beta-oestradiol. The combined treatment promoted cell cycle retardation in KPL-3C cells, which is associated with an upregulation of p21 by fulvestrant and gefitinib, respectively. Apoptosis was associated with downregulation of Bcl-2 by gefitinib in MDA-MB-231 cells. These results suggest an additive interaction between the EGFR-TKI gefitinib and the antioestrogen fulvestrant in ER-positive breast cancer cells.

Effects of three-month oral supplementation of beta-carotene and vitamin C on serum concentrations of carotenoids and vitamins in middle-aged subjects: a pilot study for a randomized controlled trial to prevent gastric cancer in high-risk Japanese population.

Prior to a randomized controlled trial to prevent gastric cancer by oral supplementation of beta-carotene and vitamin C in a high-risk Japanese population, we examined the serum response to three-month oral supplementation of beta-carotene (0, 3, 30 mg / day) and vitamin C (0, 50, 1000 mg / day) by a three-by-three factorial design using 54 subjects (age range = 40 - 69 years). Serum concentrations of carotenoids, alpha-tocopherol, and ascorbic acid were examined at baseline, and one, two, and three-month points. Both serum beta-carotene and ascorbic acid were significantly higher in high-dose groups than in each placebo group during the supplementation. The serum beta-carotene increased gradually (597 - 830% increase) during the study, whereas the serum ascorbic acid reached nearly a steady-state at the one-month point and remained stable thereafter (88 - 95% increase). No statistically significant interaction between beta-carotene and vitamin C supplementations was observed either for serum beta-carotene or for serum ascorbic acid. Among carotenoids and alpha-tocopherol examined, serum lycopene in the high-dose beta-carotene group was significantly higher than in the placebo group at all points. No unfavorable change in carotenoids and alpha-tocopherol was observed in any group.

Hereditary spastic paraplegia with a thin corpus callosum and thalamic involvement in Japan.

The authors examined two Japanese siblings with a recessive hereditary spastic paraplegia (HSP) with dementia and a thin corpus callosum. Both showed thalamic glucose hypometabolism on PET. Recessive HSP with a thin corpus callosum is a rare disorder, with less than 20 reported patients, that may be a Japanese subtype of HSP.

[Bactericidal and anti-toxin activities of catechin on enterohemorrhagic Escherichia coli].

We examined the bactericidal activity of catechin, an astringent ingredient of tea, on enterohemorrhagic Escherichia coli (EHEC) O157:H7 and the anti-toxin activity of catechin on vero toxin (VT), the main pathogenic factor of EHEC O157:H7. To examine bactericidal activity, we added 1 X 10(4) CFU/ml bacteria to 1.25 to 20 W/V% of green tea extract or the PBS solution containing 25 to 400 micrograms/ml of (-) epigallocatechin gallate (EGCg), which is the main catechin ingredient of green tea leaf, and counted the number of live bacteria at various intervals. After 3 to 5 hours, no live bacteria were seen in 1.25 to 2.5 (regular drinking concentration) % green tea extract. In the high concentrations of 100 to 400 micrograms/ml EGCg the number of live bacteria decreased with time and after 24 hours no survivors were seen. In the low concentrations of 25 to 50 micrograms/ml EGCg, however, no change was observed in the number of live bacteria during 5 hours. After 24 hours the bacteria in 50 micrograms/ml were killed and the number of bacteria in 25 micrograms/ml decreased to one tenth of that at the start. To examine the anti-toxin activity, we mixed equal volumes of 2 ng/0.1 ml VT2 and 0.5 to 2 mg/0.1 ml catechin in vitro and incubated them at 37 degrees C for various times. Then we inoculated 0.2 ml of the mixture intraperitonealy to BALB/c mice. One mg of catechin inhibited by 100% the lethal toxicity of 2 ng of VT2 (LD 100) to mice. The inhibition of lethal toxicity of VT2 by catechin depended on the incubation time. The rate of inhibition was 0, 40 and 100% for 9, 12 and 18-24 hours incubation, respectively. These results suggest that catechin has not only bactericidal activity on EHEC O157:H7 but also anti-toxin activity on vero toxin.

A randomized controlled trial for chemoprevention of gastric cancer in high-risk Japanese population; study design, feasibility and protocol modification.

We have initiated a population-based, double-blind, randomized controlled trial to examine the effects of supplementation of beta-carotene and vitamin C on the incidence of gastric cancer. The subjects were participants in an annual health screening program conducted by four municipalities in Akita prefecture, one of the regions with the highest mortality from gastric cancer in Japan. We measured their serum levels of pepsinogens (PGs) I and II, and asked persons diagnosed with chronic atrophic gastritis (defined as PG I < 70 ng/ml and PG I/PG II ratio < 3.0) to take diet supplements containing 0 or 15 mg/day beta-carotene and 50 or 500 mg/day vitamin C for 5 years. During the first year of recruitment conducted in one village from June through September, 1995, 52% (635/1214) of screening participants had chronic atrophic gastritis and 73% (439/602) of eligible persons responded. However, in response to a National Cancer Institute press report released on January 18, 1996, indicating that two beta-carotene trials had shown no benefit and potential harm from the supplement, we discontinued the beta-carotene and continued with the trial using only vitamin C. Of 397 participants remaining at this point, 77% (305) consented to stay in the study. The results indicate that a randomized controlled trial for cancer prevention is feasible in the Japanese asymptomatic population.

A pilot study for a randomized controlled trial to prevent gastric cancer in high-risk Japanese population: study design and feasibility evaluation.

Observational epidemiological studies suggest that some nutrients reduce the risk of gastric cancer and that individuals with atrophic gastritis are at high risk of developing gastric cancer. One possible measure for gastric cancer prevention is therefore nutritional supplementation for the high risk group. Before recommending this strategy for the general public, however, a randomized controlled trial (RCT) is necessary. To evaluate the feasibility of an RCT, the authors conducted a pilot study using recipients of a health check-up program in a general hospital in Japan. The subjects who were asked to participate in the trial had been diagnosed as having atrophic gastritis on the basis of serum pepsinogen I < 70 ng/ml and the ratio of pepsinogen I to II < 3.0. They were requested to ingest double-blinded capsules containing different levels of vitamin C and beta-carotene every day. Out of the 219 subjects (118 males, 101 females) who were eligible for the study and had the required pepsinogen measurement, 90 (41%) met the criteria for atrophic gastritis. Among them, 55 (61%) (35 males, 20 females) gave their informed consent to participate in the RCT. Fifty-four participants completed a 3-month course of supplementation, and all of them agreed to a 5-year supplementation period. The authors concluded that an RCT using double-blinded nutritional supplements and targeting apparently healthy individuals is feasible in an intervention study for cancer prevention in Japan.

[Inhibition of the infectivity of influenza virus by black tea extract].

We determined whether black tea extract inhibits the infectivity of influenza virus to mice. When mice were inoculated intranasally with 10(5.3) PFU influenza viruses (10(1.3) LD50), their body weight decreased and all died within 10 days. Whereas, when mice were inoculated i.n. with the mixture of influenza viruses and 2% (w/w) black tea extract, 5 min after mixing, all mice showed normal body-weight increase and survived. Neutralizing antibody to influenza virus was not detected in nine of 10 survived mice. The results indicate that black tea extract at beverage concentration (2% w/w) inhibits almost completely the infectivity of influenza virus to mice and that in vivo reversion of the tea-inactivated influenza virus does not occur.

Inhibition of the infectivity of influenza virus by tea polyphenols.

(-)Epigallocatechin gallate (EGCg) and theaflavin digallate (TF3) (1-10 microM) inhibited the infectivity of both influenza A virus and influenza B virus in Madin-Darby canine kidney (MDCK) cells in vitro. Study by electron microscope revealed that EGCg and TF3 (1 mM) agglutinated influenza viruses as well as did antibody, and that they prevented the viruses from adsorbing to MDCK cells. EGCg and TF3 more weakly inhibited adsorption of the viruses to MDCK cells. EGCg and TF3 (1-16 microM) also inhibited haemagglutination by influenza viruses. These findings suggest that tea polyphenols bind to the haemagglutinin of influenza virus, inhibit its adsorption to MDCK cells, and thus block its infectivity.

[Blood concentrations of futraful, uracil and 5-fluorouracil (5-FU) after UFT administration in the various reconstructions after gastrectomy. Saitama UFT Research Group].

The study was undertaken in the total of 58 gastric cancer patients among which 17 of Billroth (BI), 14 of Billroth II (B II) anastomosis after subtotal gastrectomy, and 7 of jejunal interposition, 9 of double tract and 11 of Roux en Y anastomosis after total gastrectomy were included. Blood samples were taken before 200 mg of per oral UFT administration and after 1, 2, 3, 5 and 7hrs. consecutively. The blood Futraful (FT) level in the total gastrectomy groups reached peak concentration within 1hr and kept in relatively high level during the observation period of 7hrs. The time to maximum FT concentration delayed in almost of B I and a few of B II patients. The concentration curves of uracil (URA) and 5-FU were similar in shape, revealing steep increase and decrease except B I anastomosis which showed gentle course. The plotted maximum concentrations of URA and 5-FU in the every type of reconstruction showed a significant correlation in the regression line. In the analysis of AUC, URA/FT was under 10%, suggesting the longer retention of the unmetabolite type of FT and early disappearance of URA. The ratio of 5-FU/FT was indifferent in each reconstruction. 5-FU/URA was higher in subtotal rather than total gastrectomy groups. From the data obtained, blood concentration of 5-FU after UFT administration was considered to depend on the emptying status in the gastrectomies. And moreover, it depended on blood URA level, since FT from which 5-FU was derived, was kept still sufficiently remained during observation period.

Mitogenic activity of (-)epigallocatechin gallate on B-cells and investigation of its structure-function relationship.

(-)Epigallocatechin gallate (EGCg), the main constituent of green tea, strongly enhanced the direct plaque-forming cell (PFC) response to sheep red blood cells (SRBC) in vitro and showed strong mitogenic activity for mouse splenic B-cells but not for splenic T-cells and thymocytes. The enhancement of B-cell proliferation was not mediated by macrophages since their removal did not eliminate the activity. Among the derivatives of catechin examined, (+)catechin (C); (-)epicatechin (EC); (-)-epigallocatechin (EGC); (-)epicatechin gallate (ECg); (-)epigallocatechin gallate (EGCg); and theaflavin digallate (TF3), only the derivatives with the galloyl group (ECg, EGCg, and TF3) displayed significant enhancement of the spontaneous proliferation of B-cells. Structural analogs of the catechin and galloyl groups were also examined in the system. Gallic acid and tannic acid induced some enhancement, but rutin, pyrogallol and caffeine did not. The results indicate that the galloyl group on EGCg was responsible for enhancement. However, the basic conformations of the catechins are also important, because ECg, EGCg, TF3, gallic acid, and tannic acid had quite different potencies to induce B-cell proliferation.

[Protective activity of tea and catechins against Bordetella pertussis].

We examined the bactericidal activity of tea and catechins against Bordetella pertussis. Green tea, black tea and coffee showed marked bactericidal activity at their concentrations in beverages, while pu-erh tea killed the bacteria in a moderate way. (-) Epigallocatechin gallate (EGCg) and theaflavin digallate (TF3) showed also marked bactericidal activity. Green tea and black tea also effectively blocked the adhesion of B. pertussis to HeLa and CHO cells, whereas ECGg and TF3 could not. EGCg and TF3 markedly inactivated leuco-lymphocytosis promoting activity of pertussis toxin. Black tea showed slight but significant inactivation of the activity, whereas green tea showed no inactivation. These results suggest that green tea, black tea, EGCg and TF3 might act as prophylactic agents against pertussis infection.

[Antimicrobial and microbicidal activities of tea and catechins against Mycoplasma].

We examined tea extracts, (-) epigallocatechin gallate (EGCg) and theaflavin digallate (TF3) for their antimicrobial and microbicidal activities against Mycoplasma. Green tea and black tea showed antimicrobial activities against M. pneumoniae. At a concentration of 0.2% green tea and black tea showed microbicidal activities against M. pneumoniae and M. orale but not against M. salivarium. Extracts of pu-erh tea showed a slight microbicidal activity against M. pneumoniae and M. orale. EGCg purified from green tea and TF3 from black tea markedly showed microbicidal activities against M. pneumoniae. M. orale and M. salivarium. These results suggest that tea and catechins can be used as prophylactic agents against Mycoplasma pneumoniae infection.

The protective activity of tea catechins against experimental infection by Vibrio cholerae O1.

Tea catechins inhibited the fluid accumulation induced by cholera toxin in sealed adult mice. The catechins also reduced fluid accumulation by Vibrio cholerae O1 in ligated intestinal loops of rabbits. These findings suggest that tea catechins may possess protective activity against V. cholerae O1.

[Antibacterial and bactericidal activities of tea extracts and catechins against methicillin resistant Staphylococcus aureus].

We examined tea extract, (-) epigallocatechin gallate (EGCg) and theaflavin digallate (TF3) for their antibacterial and bactericidal activities against methicillin resistant Staphylococcus aureus (MRSA) and food poisoning strains of S. aureus. Twenty percent tea extract (50 microliters), EGCg (63 micrograms) and TF3 (125 micrograms) added to one ml of culture medium each inhibited the growth of all strains of MRSA and food poisoning S. aureus tested. Tea extract showed also a bactericidal activity against MRSA even at the same concentration of as in ordinarily brewed tea. EGCg at a concentration of 250 micrograms/ml showed a bactericidal activity against MRSA but not against food poisoning S. aureus, but at 500 micrograms/ml reduced markedly the viable number within 48h. These results suggest that tea and catechin can be used as prophylactic agents against MRSA infection.

[Antifungal and fungicidal activities of tea extract and catechin against Trichophyton].

We examined tea extract, (-) epigallocatechin gallate (EGCg) and theaflavin digallate (TF3) for their antifungal and fungicidal activities against Trichophyton mentagrophytes, T. rubrum, Candida albicans and Cryptococcus neoformans. Tea extract (2.5%) inhibited completely the growth of both T. mentagrophytes and T. rubrum. EGCg at 2.5 mg/ml failed to inhibit their growth, whereas TF3 at 0.5 mg/ml inhibited the growth. EGCg (1mg/ml) showed no fungicidal activity against Trichophyton. TF3 (1mg/ml) killed Trichophyton by a long time contact (72-96 hrs). Tea extract showed a fungicidal activity against Trichophyton in a dose- and contact time-dependent manner. It did not inhibit the growth of C. albicans, but at a high concentration, inhibited slightly the growth of C. neoformans. It had no fungicidal activity against C. albicans or C. neoformans.

The protective activity of tea against infection by Vibrio cholerae O1.

Extracts of black tea exhibited bactericidal activity against Vibrio cholerae O1. The tea extract inhibited the haemolysin activity of V. cholerae O1, El Tor and the morphological changes of Chinese hamster ovary cells induced by cholera toxin. Tea extract also reduced fluid accumulation induced by cholera toxin in sealed adult mice and by V. cholerae O1 in ligated intestinal loops of rabbits. These findings suggest that tea has protective activity against V. cholerae O1.

[Antibacterial and anti-hemolysin activities of tea catechins and their structural relatives].

Among catechins tested, (-)epigallocatechin (EGC), (-)epicatechin gallate (ECg), (-) epigallocatechin gallate (EGCg) inhibited the growth of Staphylococcus aureus, Vibrio cholerae O1 classical Inaba 569B and El Tor Inaba V86. S. aureus was more sensitive than V. cholerae O1 to these compounds. EGCg showed also a bactericidal activity against V. cholerae O1 569B. Pyrogallol showed a stronger antibacterial activity against S. aureus and V. cholerae O1 than tannic and gallic acid. Rutin or caffein had no effect on them. ECg and EGCg showed the most potent anti-hemolysin activity against S. aureus alpha-toxin, Vibrio parahaemolyticus thermostable direct hemolysin (Vp-TDH) and cholera hemolysin. Among catechin relatives, only tannic acid had a potent anti-hemolysin activity against alpha-toxin. These results suggest that the catechol and pyrogallol groups are responsible for the antibacterial and bactericidal activities, while the conformation of catechins might play an important role in the anti-hemolysin activity.

[Antibacterial and bactericidal activities of Japanese green tea].

We found that extracts of Japanese green tea leaves inhibited the growth of various bacteria causing diarrheal diseases. All tea samples tested showed antibacterial activity against Staphylococcus aureus, S. epidermidis, Vibrio cholerae O1, V. cholerae non O1. V. parahaemolyticus, V. mimicus, Campylobacter jejuni and Plesiomonas shigelloides. None of the tea samples had any effect on the growth of V. fluvialis, Aeromonas sobria, A. hydrophila, Pseudomonas aeruginosa, Salmonella enteritidis, enteroinvasive Escherichia coli, enterohemorrhagic E. coli, enteropathogenic E. coli, enterotoxigenic E. coli, Enterobacter cloacae or Yersinia enterocolitica. Salmonella and Shigella showed susceptibilities different depending on the kind of Japanese green tea. Japanese green tea showed also bactericidal activity over S. aureus, V. parahaemolyticus and even enteropathogenic E. coli which was not sensitive when tested by cup method. The bactericidal activity was shown even at the drinking concentration in daily life.