Hydrogen-rich bath with nano-sized bubbles improves antioxidant capacity based on oxygen radical absorbing and inflammation levels in human serum

This study compared the effects of hydrogen-water (HW) bath on the oxygen radical absorption-based antioxidant capacity and the inflammatory indicator, C-reactive protein (CRP), in serum between healthy volunteers and inflammatory/collagen disease-patients. The HW bath apparatus supplied nano-bubbles with a diameter of 110 ± 10 nm and 338-682 μg/L of dissolved hydrogen after 120 minutes electrolysis, and nano-bubbles increased to 9.91 × 107/mL along with the increase of correlative dissolved hydrogen. Ten-minute HW bath increased the oxygen radical absorption-based antioxidant capacity to 110.9 ± 9.2% at post-bathing 120 minutes, although unaltered with 10-minute normal water bath at 40°C in healthy subjects. The CRP level was repressed to 70.2 ± 12.1% at 120 minutes after HW bath, although rather increased for normal water bath. In the patients with connective tissue diseases, the CRP level was repressed to 3-24% upon 9 days to 4 months of HW bathing. In another six patients with diverse autoimmune-related diseases, upon daily HW bathing as long as 2-25 months, the pre-bathing CRP level of 5.31 mg/dL decreased to 0.24 mg/dL being within the standard-range, with relief of visible inflammatory symptoms for some cases. Thus, the HW bath with high-density nano-bubbles has beneficial effects on serum antioxidant capacity, inflammation, and the skin appearance. The study was approved by the Committee of Ethics, Japanese Center of Anti-Aging Medical Sciences (Authorization No. H-15-03-2, on January 15, 2019), which was a non-profitable organization officially authenticated by the Hiroshima Prefecture Government of Japan. (

Neutral pH Hydrogen-Enriched Electrolyzed Water Achieves Tumor-Preferential Clonal Growth Inhibition Over Normal Cells and Tumor Invasion Inhibition Concurrently With Intracellular Oxidant Repression

The properties and effects of neutral pH hydrogen-enriched electrolyzed water (NHE water) on tumor cells were examined. NHE water diminished hydroxyl radicals as demonstrated by ESR in a cell-free system. Human tongue carcinoma cells HSC-4 were inhibited for either colony formation efficiencies or colony sizes by NHE water without significant inhibition to normal human tongue epithelial-like cells DOK. Furthermore, NHE water caused growth inhibition, cell degeneration, and inhibition of invasion through the reconstituted basement membrane to human fibrosarcoma cells HT-1080. Intracellular oxidants such as hydroperoxides and hydrogen peroxides were scavenged in HSC-4 or HT-1080 cells by NHE water. In the human oral cavity, a dissolved hydrogen concentrations (DH) of NHE water was drastically declined from 1.1 to 0.5 ppm, but settled to 0.3-0.4 ppm until 180 s, upon static holding without gargling. Thus, NHE water was shown to achieve tumor-preferential growth inhibition and tumor invasion together with scavenging of intracellular oxidants, and is expected as a preventive material against tumor progression and invasion.

Hydrogen-rich water achieves cytoprotection from oxidative stress injury in human gingival fibroblasts in culture or 3D-tissue equivalents, and wound-healing promotion, together with ROS-scavenging and relief from glutathione diminishment

The aim of the present study is to investigate protective effects of hydrogen-rich water (HW) against reactive oxygen species (ROS)-induced cellular harmful events and cell death in human gingival fibroblasts (HGF) and three-dimensional (3D-) gingival tissue equivalents. HW was prepared with a magnesium stick in 600-mL double distilled water (DDW) overnight. Dissolved hydrogen was about 1460 ± 50 μg/L versus approximately 1600 μg/L for the saturated hydrogen. Under cell-free conditions, HW, dose-dependently, significantly scavenged peroxyl radicals (ROO·) derived from 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH). Extract from HW-treated HGF cells scavenged ROO· more markedly than that from DDW-treated cells, suggesting that HW can increase the intracellular antioxidant capacity. Hydrogen peroxide dose-dependently increased the intracellular ROS generation, which was significantly repressed by HW, both in the cytoplasm and nuclei. LIVE/DEAD staining and our original cell viability dye-extraction assay showed that HW significantly protected HGF cells from hydrogen peroxide-induced cell death. Hydrogen peroxide also diminished the contents of intracellular glutathione, which were appreciably relieved by HW-pretreatment. Additionally, HW noticeably prevented cumene hydroperoxide-induced generation of cellular ROS in epidermis parts of 3D-gingival equivalents. The in vitro scratch assay showed that HW was able to diminish physical injury-induced ROS generation and promote wound healing in HGF cell monolayer sheets. In summary, HW was able to increase intracellular antioxidative capacity and to protect cells and tissue from oxidative damage. Thus, HW might be used for prevention/treatment of oxidative stress-related diseases.

Hydrogen Nano-Bubble Water Suppresses ROS Generation, Adipogenesis, and Interleukin-6 Secretion in Hydrogen-Peroxide- or PMA-Stimulated Adipocytes and Three-Dimensional Subcutaneous Adipose Equivalents

Reactive oxygen species (ROS)-induced oxidative stress in adipose tissue is associated with inflammation and the development of obesity-related metabolic disorders. The aim of this study is to investigate the effects of hydrogen nano-bubble water (HW) on ROS generation, adipogenesis, and interleukin-6 (IL-6) secretion in hydrogen peroxide (H2O2) or phorbol 12-myristate 13-acetate (PMA)-stimulated OP9 adipocytes, and three-dimensional (3D) subcutaneous adipose equivalents. Nanoparticle tracking analysis showed that fresh HW contains 1.17 × 108/mL of nano-sized hydrogen bubbles. Even after 8 to 13 months of storage, approximately half of the bubbles still remained in the water. CellROX® staining showed that HW could diminish H2O2- or PMA-induced intracellular ROS generation in human keratinocytes HaCaT and OP9 cells. We discovered that PMA could markedly increase lipid accumulation to 180% and IL-6 secretion 2.7-fold in OP9 adipocytes. Similarly, H2O2 (5 µM) also significantly stimulated lipid accumulation in OP9 cells and the 3D adipose equivalents. HW treatment significantly repressed H2O2- or PMA-induced lipid accumulation and IL-6 secretion in OP9 adipocytes and the 3D adipose equivalents. In conclusion, HW showed a possibility of repressing oxidative stress, inflammatory response, and adipogenesis at cellular/tissue levels. It can be used for preventing the development of metabolic disorders amongst obese people.