Active oxygen species or free radicals are considered to cause extensive oxidative damage to biological macromolecules, which brings about a variety of diseases as well as aging. The ideal scavenger for active oxygen should be ‘active hydrogen’. ‘Active hydrogen’ can be produced in reduced water near the cathode during electrolysis of water. Reduced water exhibits high pH, low dissolved oxygen (DO), extremely high dissolved molecular hydrogen (DH), and extremely negative redox potential (RP) values. Strongly electrolyzed-reduced water, as well as ascorbic acid, (+)-catechin and tannic acid, completely scavenged O.-2 produced by the hypoxanthine-xanthine oxidase (HX-XOD) system in sodium phosphate buffer (pH 7.0). The superoxide dismutase (SOD)-like activity of reduced water is stable at 4 degrees C for over a month and was not lost even after neutralization, repeated freezing and melting, deflation with sonication, vigorous mixing, boiling, repeated filtration, or closed autoclaving, but was lost by opened autoclaving or by closed autoclaving in the presence of tungsten trioxide which efficiently adsorbs active atomic hydrogen. Water bubbled with hydrogen gas exhibited low DO, extremely high DH and extremely low RP values, as does reduced water, but it has no SOD-like activity. These results suggest that the SOD-like activity of reduced water is not due to the dissolved molecular hydrogen but due to the dissolved atomic hydrogen (active hydrogen). Although SOD accumulated H2O2 when added to the HX-XOD system, reduced water decreased the amount of H2O2 produced by XOD. Reduced water, as well as catalase and ascorbic acid, could directly scavenge H2O2. Reduce water suppresses single-strand breakage of DNA b active oxygen species produced by the Cu(II)-catalyzed oxidation of ascorbic acid in a dose-dependent manner, suggesting that reduced water can scavenge not only O2.- and H2O2, but also 1O2 and .OH.
Active oxygen species are considered to cause extensive oxidative damage to biological macromolecules, which bring about a variety of diseases as well as aging. Reduced water produced near cathode during electrolysis of water exhibits high pH, low dissolved oxygen, extremely high dissolvedmolecular hydrogen, and extremely negative redox potential values. Recently we found that strongly electrolyzed reduced water scavenges active oxygen species and protects DNA from oxidative damage (Shirahata, S. et al., Biochem. Biophys. Res. Commun., 234, 269–274 (1997)). Electrolyzed reduced water suppressed the growth of human normal fibroblast TIG-1, human lung adenocarcinoma A549, and human uterine cervix cancer HeLa, indicating that reduced water affects the signaling pathway of cell cycle. The expression of the interleukin-6 gene was enhanced by reduced water as well as ascorbic acid, (+)-catechin and tannic acid when added to the culture of human osteosarcoma MG-63 cells, suggesting that reduced water acts as a reductant to cells.
In the type 2 diabetes, it has become clear that reactive oxygen species (ROS) cause reduction of glucose uptake by inhibiting the insulin-signaling pathway in muscle cells and adipocytes. We demonstrated that electrolyzed-reduced water (ERW) scavenges ROS and protects DNA from oxidative damage1). Here we found that ERW scavenges ROS in insulin-responsive L6 myotubes and mouse3T3/L1 adipocytes. Uptake of 1-deoxy-D- glucose (2-DOG) into both L6 cells and 3T3/L1 cells was stimulated by ERW in the presence or absence of insulin. This insulin-like activity of ERW was mediated by the activation of PI-3 kinase, resulting in stimulation of translocation of glucose transporter GLUT4 from microsome to plasma membrane. These results suggest that ERW may be useful to improve insulin-independent type 2 diabetes.
Electrolyzed reduced water and natural waters such as Hita Tenryosui water in Japan, Nordenau water in Germany and Tracote water in Mexico, which are known to improve various diseases, were all and- oxidative waters which could scavenge intracellular reactive oxygen species. The and-oxidative waters stimulated not only the glucose uptake of rat myotube L6 cells, but also the secretion of insulin from a pancreatic beta cell line HIT-T15. The anti-oxidative waters improved the damage in the sugar tolerance test of type 2 diabetes model mice (db/db mice). A clinical investigation demonstrated that Nordenau water could significantly improve the diabetes mellitus.
Electrolyzed-reduced water (ERW) produced by electrolyzing water in cathode side has the ability to scavenge reactive oxygen species (ROS). Here, we investigated the effect of ERW on oxidative stress-induced neural cell death by glutamate. When cell viability assay was performed using primary rat cerebral cortical culture as neural model, ERW suppressed neural cell death by glutamate. Furthermore, intracellular ROS levels were reduced by ERW, suggesting that suppressive effect of ERW on the glutamate-induced neural cell death was due to the suppression of glutamate-induced ROS augmentation by ERW.
We examined the suppressive effect on lipid peroxidation of electrolyzed reduced water (ERW), which exhibited reactive oxygen species (ROS)-scavenging activity in vitro and in cultured cells. The oxidation of linoleic acid by 2, 2’-azobis (2-amidinopropane) hydrochloride (AAPH) was suppressed by ERW in vitro. ERW also suppressed the lipid peroxidation in cell membrane of mouse lymphoblast YAC-1 cells. Transparent electron microscopic analysis revealed that platinum in ERW existed as a colloidal form of nm size. Concentrated ERW suppressed the lipid peroxidation in a manner dependent upon the concentration of platinum nanocolloids, and chemical synthesized platinum nanocolloids exhibited a similar suppressive effect on lipid peroxidation to that of ERW, suggesting that platinum nanocolloids contained in ERW were reducing agents to suppress the lipid peroxidation.
We have proposed an active hydrogen reduced water theory that active hydrogen produced by electrolysis of water is stabilized in the form of hydrogenated metal nanocolloids in electrolyzed reduced water (ERW) and scavenges intracellular reactive oxygen species (ROS). Because various brain diseases are caused by oxygen stress, we examined the effect of ERW on oxidative stress-induced apoptois of neuronal cells. ERW suppressed the H2O2-induced cell death of mouse neuroblastoma N1E115 cells, rat pheochromocytoma PC12 cells and mouse neuronal stem SFME cells. ERW lowered the intracellular ROS level of N1E115 cells, suppressing the H2O2-induced decrease of mitochondrial membrane potential and intracellular ATP level, which are markers of apoptosis. These results suggested the effectiveness of ERW for prevention of various brain diseases caused by oxidative stress.
Recent studies have demonstrated that reactive oxygen species9ROS9and the resulting oxidative stress play an important role in apoptosis. Apoptosis is implicated in pathophysiology of diabetes mellitus. Antioxidants can block or delay apoptosis. We have demonstrated that reduced water (RW) such as hydrogen-rich electrolysed-reduced water (ERW) and natural reduced waters (NRW) like Hita Tenryosui water in Japan and Nordenau water in Germany could scavenge ROS and stimulate glucose intake into muscle and adipocytes. This study investigated the effect of reduced water9RW9on oxygen radicals and apoptosis of pancreatic β -cells by alloxan. Incubation of HIT-T15 cells with alloxan, a diabetogenic compound, resulted in the increased intracellular ROS level, a decrease in viability of cells, the formation of DNA fragmentation and Sub-G1 phase. The generation of ROS, the formation of DNA fragmentation and Sub-G1 phase, the lowering of cell viability by alloxan toxicity can be suppressed by treatment with RW. In contrast, HIT-T15 cells treated with Mineral water were not observed. These results suggest that RW protected pancreatic β-cell from the alloxan-induced apoptosis by preventing the alloxan-derived oxygen radical generation.
Chronic hemodialysis (HD) patients increase erythrocyte susceptibility to hemolysis and impair cell survival. We explored whether electrolyte-reduced water (ERW) could palliate HD-evoked erythrocyte impairment and anemia. Forty-three patients undergoing chronic HD were enrolled and received ERW administration for 6 month. We evaluated oxidative stress in blood and plasma, erythrocyte methemoglobin (metHb)/ferricyanide reductase activity, plasma metHb, and proinflammatory cytokines in the chronic HD patients without treatment (n=15) or with vitamin C (VC)- (n=15), vitamin E (VE)-coated dialyzer (n=15), or ERW treatment (n=15) during an HD course. The patients showed marked increases (15-fold) in blood reactive oxygen species, mostly H(2)O(2), after HD without any treatment. HD resulted in decreased plasma VC, total antioxidant status, and erythrocyte metHb/ferricyanide reductase activity and increased erythrocyte levels of phosphatidylcholine hydroperoxide (PCOOH) and plasma metHb. Antioxidants treatment significantly palliated single HD course-induced oxidative stress, plasma and RBC PCOOH, and plasma metHb levels, and preserved erythrocyte metHb /ferricyanide reductase activity in an order VC>ERW>VE-coated dialyzer. However, ERW had no side effects of oxalate accumulation easily induced by VC. Six-month ERW treatment increased hematocrit and attenuated proinflammatory cytokines profile in the HD patients. In conclusion, ERW treatment administration is effective in palliating HD-evoked oxidative stress, as indicated by lipid peroxidation, hemolysis, and overexpression of proinflammatory cytokines in HD patients.
The analysis using the DBNBS reduction method and the DCFH-DA intracellular reactive oxygen species (ROS) determination method revealed that ERW and diseases-improvable natural waters such as Nordenau water in Germany and Hita water in Japan were all reduced waters (RWs) which could function as active hydrogen donors and intracellular ROS scavengers. RWs suppressed the activity of protein tyrosine phosphatase (PTP), which inactivates insulin receptor, suggesting their anti-type 2 diabetes effects via redox regulation. The clinical test of 356 diabetes patients drinking Nordenau water in Germany resulted in the improvement of the relevant tests parameters after 6 days, suggesting the correlation of these changes with the fluctuation of ROS levels in their blood.