Scientific Studies


H2-rich water as a green antioxidant was applied for deoxygenation of graphene oxide (GO) suspensions. The ability of H2-rich water for deoxygenation of GO sheets was found comparable to the ability of hydrazine (as a standard and powerful reductant), using X-ray photoelectron spectroscopy. In fact, the O/C ratio of GO sheets could be reduced from 0.51 to 0.21 and 0.16 by H2-rich water and hydrazine, respectively. More importantly, while C–N bond formation is one of the side effects of GO reduction by hydrazine, no chemical C–N bond was found on the H2-water-reduced GO (rGO) sheets. This also resulted in a better restoration of the graphitic structure of the H2-water-rGO, as confirmed by Raman spectroscopy. Although H2-rich water exhibited slightly lower deoxygenation efficiency than hydrazine, the absence of any C–N bond on the H2-water-rGO resulted in an excellent electrical conductivity (corresponding to the sharp reduction in the electrical sheet resistance (Rs) of the GO sheets from ∼6.3 × 1010 to 7.2 × 105 Ω/sq) which is comparable with the typical conductivity of hydrazine-rGO sheets (here, with Rs value of ∼4.4 × 105 Ω/sq). These results suggest application of H2-rich water as an effective substitute for hydrazine in environment-friendly and mass production of rGO sheets.

Several of the procedures necessary for cryopreservation of excised zygotic embryonic axes are known to be accompanied by emission of damaging levels of reactive oxygen species (ROS). These have been shown to be associated with shoot apical meristem necrosis, curtailing subsequent axis development to root production only, particularly for tropical/subtropical recalcitrant-seeded species. Here we report on the application of the principles of electrochemistry in the generation of strongly reducing, high pH cathodic water, by electrolysis of a solution containing calcium and magnesium chloride. The cathodic water in which Strychnos gerrardii axes were immersed for 30 min following dehydration, and importantly, after dehydration followed by cryopreservation, was shown to have strongly antioxidative properties in counteracting the damaging effects of ROS bursts and promoting shoot development. In a parallel experiment, axes of Boophane disticha exhibited enhanced total antioxidant activity when exposed to cathodic water both immediately following excision, and after flash-drying. For both species, the efficacious effects of cathodic treatment were manifest after the axes had been in culture for 4 h, suggesting that ROS were not quenched at source, but probably counteracted by enhancement of activity of endogenous antioxidants. Cathodic water therefore affords a non-toxic means of amelioration of oxidative, stress-related damage, which, coupled with the strongly fungicidal activity of the acidic, anionic water fraction, offers significant, and apparently non-injurious, advances towards successful cryopreservation of germplasm – and probably generally improved success of in-vitro-based procedures for plant tissues.

Antioxidant vitamins and enzymes such as superoxide dismutase, catalase and glutathione peroxidase are considered to function as scavengers against reactive oxygen species and to provide protection against reactive oxygen species, including free radicals. Although antioxidants such as L-ascorbic acid, d-catechin and quercetin dehydrate show superoxide dismutation activity, using reduced water produced in the cathode side by electrolysis as a solvent instead of 2 mM NaC1 solution of the same pH level as the reduced water increased the superoxide dismutation activity of these antioxidants. Moreover, neither the reduced water nor its electrolyte solution showed any superoxide dismutation activity by itself. On the other hand, the reduced water was able to decrease hydrogen peroxide levels. It has been found that the behaviour of H2 in reduced water, which was activated by a platinum electrode, differed from that of H2 introduced by bubbling of hydrogen gas. The former decreased H2O2, whereas the latter did not. These results suggest strongly that the increase in superoxide dismutation activity, with a proton donor such as L-ascorbic acid, is due to an increase in the dissociation activity of water while the scavenging activity for H2O2 is due to activated dissolved H2 in the reduced water.

We reported that reduced water produced by electrolysis enhanced the antioxidant effects of proton donors such as ascorbic acid (AsA) in a previous paper. We also demonstrated that reduced water produced by electrolysis of 2 mM NaCl solutions did not show antioxidant effects by itself. We reasoned that the enhancement of antioxidant effects may be due to the increase of the ionic product of water as solvent. The ionic product of water (pKw) was estimated by measurements of pH and by a neutralization titration method. As an indicator of oxidative damage, Reactive Oxygen Species- (ROS) mediated DNA strand breaks were measured by the conversion of supercoiled phiX-174 RF I double-strand DNA to open and linear forms. Reduced water had a tendency to suppress single-strand breakage of DNA induced by reactive oxygen species produced by H2O2/Cu (II) and HQ/Cu (II) systems. The enhancement of superoxide anion radical dismutation activity can be explained by changes in the ionic product of water in the reduced water.

We studied the in vitro antioxidant activities of neutral aqueous solution systems (water products marketed as drinks) containing hydrogen gas (H2), 2-carboxyethyl germanium sesquioxide (Ge-132), and platinum (Pt) nanocolloid as additives. We evaluated the abilities of these aqueous solutions to inhibit the oxidation of biomolecules catalyzed by an enzyme and induced by reactive oxygen species (ROS) and also to scavenge ROS directly using electron spin resonance (ESR) spectrometry. The concentrations of inorganic elements including Ge and Pt were measured by inductively coupled plasma-mass spectrometry (ICP-MS) and inductively coupled plasma-atomic emission spectrometry (ICP-AES). All the water products examined more or less inhibited the oxidation of 3,4-dihydroxyphenylalanine by tyrosinase and that of L-histidine in an L-ascorbic acid/Cu2+ reaction system. The results of ICP-MS and ICP-AES analyses revealed that Ge, Pt, and some major minerals existed in the water products at concentrations approximately equivalent to those reported by their manufacturers. The ESR spectra indicated that the dissolved Ge-132 molecules and the supplemented Pt nanocolloid particles reduced hydroxyl and superoxide anion radicals. However, under the conditions employed, aqueous H2 did not display such a scavenging ability for these ROS. Our results suggest that H2, Ge-132 and Pt nanocolloid dissolved or supplemented in neutral aqueous media exhibited antioxidant activities in vitro due to the direct scavenging of ROS and/or by other mechanisms.