Jhs 50(5)456-465

Journal of Health Science, 50(5) 456–465 (2004)
Studies on the Properties and Real Existence of
Aqueous Solution Systems that are Assumed
to Have Antioxidant Activities by the Action of
“Active Hydrogen”

Atsushi Hiraoka,*, a Masumi Takemoto,a Takahiro Suzuki,a Atsuko Shinohara,b
Momoko Chiba,b Mika Shirao,c and Yoshihiro Yoshimurad
aDepartment of Pathological Biochemistry, Kyorin University School of Health Sciences, 476 Miyashita-cho, Hachioji-shi, Tokyo 192–8508, Japan, bDepartment of Epidemiology and Environmental Health, Juntendo University School of Medicine, 2–1–1 Hongo, Bunkyo-ku, Tokyo 113–8421, Japan, cDepartment of Human Nutrition, Jissen Woemen’s College, 1–13–1 Shinmei, Hino-shi, Tokyo 191–0016,Japan, and dDepartment of Analytical Chemistry, Faculty of Pharmaceutical Science, Hoshi University, 2–4–41 Ebara, Shinagawa-ku, Tokyo 142–8501, Japan (Received March 2, 2004; Accepted June 9, 2004) We evaluated the properties and real existence of an electrolyzed-reduced water, which we prepared, and three commercially purchased water goods, that are advertised to have antioxidant activities by the action of “activehydrogen,” on the basis of the results of examinations for inhibitory effects on the oxidative reactions of biomolecules,quantitative analyses of the minerals, and the ESR spectral data in measurement of the scavenging ability for reac-tive oxygen species. The results suggested that all of the examined aqueous solution systems undoubtedly haveantioxidant activities in vitro and that such effects are derived from ordinary molecular hydrogen (hydrogen gas)and/or (a) reductive vanadium ion(s). “Active hydrogen” seems to be absent as an effective component of the anti-oxidant activities of these aqueous solution systems.
Key words —–— reduced water, antioxidant activity, oxygen-radical scavenger, ESR spectrometry, hydrogen,
caused by an L-ascorbic acid (AsA)-Cu2+ reactionsystem.1) In that paper they also presented a hypoth- In recent years, many companies have developed esis that “active hydrogen,” namely atomic state and sold water goods (aqueous solution systems) for hydrogen (not H but H), may be contained stably in health. Some of such solutions are advertised to have the ERW.1) Shirahata also declared elsewhere that antioxidant activities scavenging harmful reactive “active hydrogen” may be present in various artifi- oxygen species (ROS). It is known that electrolysis cial and natural aqueous solution systems with anti- of aqueous solutions of electrolytes affords acidic oxidant activities.2,3) Shirahata et al. also investigated solutions near the anode (electrolyzed-oxidized wa- the biological effects of such aqueous solution sys- ters, EOWs) and alkaline solutions near the cathode tems called reduced waters. Suppression of growth (electrolyzed-reduced waters, ERWs). Shirahata et of cultured human cancer cells and acceleration of al. studied the properties of an ERW which they pre- glucose intake by cells in diabetic rats were reported pared by electrolysis of an aqueous solution of so- as the effects of reduced waters.4) In association with dium chloride (NaCl), and reported that the exam- publication of their work, some companies, such as ined ERW showed a superoxide dismutase-like ac- Nihon Trim (Osaka, Japan), Water Institute tivity alleviating the oxidative damage of DNA (Atarashiimizunokai) (Tokyo, Japan) and HitaTenryosui Kabushikigaisha (Oita, Japan) and oth-ers, have developed and sold various water goods *To whom correspondence should be addressed: Department of for health, advertising that they contain or produce Pathological Biochemistry, Kyorin University School of Health “active hydrogen” which must be a potent scaven- Sciences, 476 Miyashita-cho, Hachioji-shi, Tokyo 192–8508,Japan. Tel.: +81-426-91-0011 (ext. 4402); Fax: +81-426-91- ger for harmful ROS [“I’m Fine,” the electrolyzed- reduced water of Nihon Trim; http://www.kangen- water.jp, Water Institute (Atarashiimizunokai); an acidic EOW with antimicroorganism activities, http://www.water-institute.org, Hita Tenryousui although an alkaline ERW can also be simulta- Kabushikigaisha; http://www.hitatenryousui.co.jp].
neously generated. Among them, only the ERW was They usually referred to the first paper of Shirahata treated in this study. An aqueous solution system et al.1) as a proof for confirmation of the presence of called “I’m Fine” (IF) as a water commercial prod- “active hydrogen” in the corresponding water goods.
uct, which is advertised to be prepared by electroly- We doubted this hypothesis since, if “active hydro- sis with a Nihon Trim apparatus of underground gen” is stably present as a solute in aqueous media, water near Mt Fuji (“I’m Fine,” the electrolyzed- conventional physical and chemical knowledge must reduced water of Nihon Trim; http://www.kangen- be essentially denied. Shirahata later proposed a water.jp), was purchased from Nihon Trim. Mineral- newly-modified theory that “active hydrogen” is ac- stick water (MSW) was prepared by extraction for cumulated on the surface of metal clusters contained 1 hr at room temperature with 1.5 l of deionized in the corresponding aqueous solution systems not water (DW) of a hydrogen promoting mineral as (a) solute(s) but as (a) nanocolloidal solid (HPM)-stick called Kasseisuisokun (Mr. “Active component(s).5) More recently, the Water Institute Hydrogen,” in Japanese) purchased from the Water expressed in the internet home page (HP) that the Institute; this procedure is the same as that in the water of their commercial product contains not “ac- protocol of the Water Institute except for the use of tive hydrogen” but molecular hydrogen which is DW instead of TW [Water Institute (Atarashii- enzymatically degraded in the human body into “ac- mizunokai); http://www.water-institute.org]. Hita tive hydrogen” reacting with ROS [Water Institute tenryosui water (HT), which is advertised to be un- (Atarashiimizunokai); http://www.water-institute.
derground water collected in Hita-shi, Oita-ken, Ja- org]. At present we have little knowledge on the pan (Hita Tenryousui Kabushikigaisha; http:// properties and real existence of reduced waters www.hitatenryousui.co.jp), was purchased from Hita (aqueous solution systems with antioxidant activi- Tenryosui Kabushikigaisha. A pooled human serum ties), that are assumed to have antioxidant activity sample was taken from normal healthy persons.
by the action of “active hydrogen.” In this study, we Examinations for Antioxidant Activities —–— In-
examined the inhibitory effects of an ERW, which hibitory effects of aqueous solution systems exam- we prepared, and three commercially-purchased ined on the oxidative reactions of biomolecules were water goods, that are advertised to have or produce measured as following (1)–(3). The experiments antioxidant activities by the action of “active hydro- were performed four times for each of (1), (2) and gen,” on the oxidative reactions of biomolecules.
Determination of their mineral components (both (1) The human serum (0.25 ml each) was diluted metal elements and anions) and measurement by to 1.0 ml by adding mixtures (0.75 ml each) of ESR spectrometry of their ability to scavenge ROS samples to be examined and DW at volume ratios of were also performed. The results are discussed in 0 : 3 (the control), 1 : 2, 2 : 1 and 3 : 0, respectively; connection with possibility of the presence of “ac- samples were ERW (an ERW we prepared), IF, MSW, HT, TW (in Hino-shi, Tokyo, Japan; the sol-vent of the original electrolyte for preparation ofERW) and an aqueous solution of caffeic acid (CFA) MATERIALS AND METHODS
as a known antioxidant with an initial concentrationof 100 µM. The reaction was started by further ad- Materials —–— An ERW was prepared by elec-
dition of an aqueous solution (50 µl each) of copper trolysis of an aqueous NaCl with an ROX-10WA sulfate (CuSO ) with an initial concentration of Electrolyzed-Water Generator (Hoshizaki Electric 210 µM. The reaction mixtures thus prepared to con- Co. Ltd., Osaka, Japan). In the electrolysis using this tain Cu2+ with a final concentration of 10 µM as an apparatus, the original electrolyte solution obtained oxidant were left at room temperature for 2 hr. Then, by dissolving 140 g of NaCl in 1 l of tap water (TW) the reaction was terminated by addition of Na- is automatically introduced into the chamber to be ethylenediaminetetraacetate with a final concentra- diluted with a solution prepared by passing TW tion of 50 µM. When the reaction was started and through ion-exchanging membranes, followed by terminated, malondialdehyde (MDA) was deter- electrolysis of the mixture in the chamber at 100 V.
mined by the thiobarbituric acid method as described These procedures are normally operated to produce previously,6) and the lipid peroxide (LPO) quantity generated in each sample was finally evaluated from mass spectrometer (a Hitachi P-7000). Anions such the difference of the MDA levels between the start as chloride (Cl–), nitrite (NO –), nitrate (NO –), phos- phate (PO 3–) and sulfate (SO 2–) were quantitatively (2) Reaction mixtures consisting of a 100 mM analyzed by ion-chromatography with a Nihon trishydroxyaminomethane buffer solution (pH 7.4) (0.2 ml each), human serum (0.2 ml each) and Measurement of the ROS Scavenging Ability by
samples to be examined (0.6 ml each) were prepared; ESR Spectrometry —–— The ESR spectra were
samples were the same as in (1) except for the use taken with a JEOL JES-RE1X using a quartz flat as a known antioxidant of the same concentration of cell designed for aqueous solution, under the condi- aqueous AsA instead of CFA. The reaction mixtures tions of the magnetic field of 336.5 mT, the power thus prepared were left at 4°C in the dark, and at of 8.0 mW, the modulation frequency of 100 kHz, 0 hr (when the reaction was started), 17 hr, 41 hr and the operating frequency of 9.456 GHz, the modula- 65 hr after the preparation, bilirubin (BR) was de- tion amplitude of 0.063 mT, the gain of 200, the time termined by the diazo method as described previ- scan of 0.5 min, and the time constant of 0.03 sec.
Dimethyl sulfoxide (50 µl) and the same volume each (3) Rutin (= quercetin-3-rhamnoglucoside, Kanto of 25 mM sodium hydroxide (NaOH) and sample Chemicals, Tokyo, Japan) was dissolved at a con- solution examined (ERW, IF, MSW, HT and distilled centration of 100 µM in solutions (1.0 ml each) that water as the reference) were mixed in a disposable were prepared by adding 100 mM phosphate buffer plastic tube, followed by addition of 5,5-dimethyl- solution (pH 6.8) (0.25 ml each) to the mixtures l-pyrroline-N-oxide (DMPO) (5 µl) and 30% hydro- (0.75 ml each) of same samples as in (2) and DW at gen peroxide (H O ) (50 µl). The reaction mixtures volume ratios of 0 : 3 (the control), 1 : 2, 2 : 1 and thus prepared were sucked into the quartz flat cell 3 : 0, respectively. The enzymatic oxidation of rutin and set in the ESR apparatus. Scanning was started was started by mixing the above-mentioned rutin so- at 10 min after the addition of H O . The signal in- lutions with an equal volume of an aqueous solution tensity of detected oxygen radical species-DMPO in which 25 units of polyphenol oxidase (PPO) adducts reached plateaus between 10 and 20 min (mushroom tyrosinase, Sigma, St. Louis, MO, after the start of the reaction. The signal height of U.S.A.) was dissolved. The reaction mixtures thus the program was calculated employing a radical ana- prepared to contain PPO as a catalyst for the oxida- lyzing program attached to the apparatus. The cal- tive reaction and rutin as the substrate were then left culation was performed for the positive signal of at 25°C without shaking. When the reaction mix- hydroxy radical (•OH)-DMPO adduct and the nega- tures were prepared and 1 hr later, a constant vol- tive signal of superoxide radical (O –)-DMPO ad- ume each of the reaction mixtures was withdrawn, duct, and the ratios of signal intensity against man- and heated at 100°C for 5 min in a stoppered-vial to ganese ion (Mn2+) as the reference were obtained.
inactivate the enzyme, followed by ultrafiltration The ability of the sample solutions to scavenge •OH with Centricon-10 miniconcentrators (Amicon Ja- and O – was expressed as relative values (%) of the pan, Tokyo, Japan) at 2000 × g and 4°C for 30 min above ratios to the reference as 100%.
to remove the enzyme proteins (only the solutes with Examinations for the Antioxidant Activities of
molecular weight (MW) below 10000 can penetrate Hydrogen Gas —–— It is well known that mo-
the membrane of this tool). Twenty µl each of the lecular hydrogen (hydrogen gas) is generated when ultrafiltrates were then injected into HPLC, by which metallic Mg is dissolved in water to form magne- rutin can be separated from its oxidation products sium cation and hydroxyl anion. A reaction mixture of Mg/DW was prepared by adding 200 mg of me- Analyses of the Minerals —–— Prior to the analy-
tallic Mg powder to 100 ml of DW, and allowed to ses, samples to be examined (ERW, IF, MSW, HT stand for 24 hr at room temperature. At 0 hr (when and TW) were passed through a filter with a pore it was prepared), 1 hr, 2 hr, 3 hr and 24 hr after the size of 0.45 µm. The concentrations of the major preparation, a constant volume each of the solution metal elements, such as Na, potassium (K), calcium was withdrawn and passed through a filter with a (Ca) and magnesium (Mg), were measured by an pore size of 0.45 µm. The filtrates (the aqueous phase atomic absorbance spectrometer (a Perkin Elmer of the reaction mixture) obtained at each time were Analyst 800). Other trace metals listed in Table 1 examined for the inhibitory effects on biomolecules, were determined by a microwave-induced plasma as in (1)–(3) descrived above, and also for the Mg Table 1. The Concentrations of Minerals in Samples Examined
Trace Metals (µg/l, by MIP-MSh)) a) Electrolyzed reduced water, b) “I’m Fine,” c) Mineral-stick water, d) Hira tenryosui water, e) Tap water, f) Atomic absorbance spectroscopy, g) Under detectable (below 0.1 mg/lfor AA and IC and 0.1 µg/ml for MIP-MS), h) Microwave-induced mass spectroscopy. Otherabbreviations in the text.
concentrations with a commercial kit of Wako Pure oxidant activities as in (1)–(3). In order to elucidate Chemicals (Osaka, Japan) (Mg Test Wako-B) which whether the antioxidant activities of these aqueous employs the colorimetric method using xylylazo- solution systems are deribed only from the low MW solutes, their ultrafiltrates, that were obtained by cen- Others —–— In order to evaluate the degree of
trifuging of these solutions as described in (3), as contribution of the substance(s) other than (a) vola- well as the control sample centrifuged under the same tile component(s), in generation of the antioxidant conditions without ultrafiltration, were also exam- activities of ERW, IF, MSW and HT, samples of these ined for the antioxidant activities as in (1)–(3). Fur- aqueous solution systems after boiled and re-cooled thermore, two aqueous solutions with approximately in an opened container were examined for the anti- the same mineral compositions as in ERW and MSW (Table 1) were prepared by dissolving 55 mg of NaCl ume ratios to DW tended to be elevated in associa- and 16 mg of NaOH in 100 ml of TW (for ERW) tion with increase in the volume ratios, indicating and by dissolving magnesium hydroxide (Mg(OH) ) that the sample involving only TW (0.83 ± in DW at the concentration of 38 mg/l (for MSW), 0.05 nmole/ml, n = 4) was significantly greater respectively. The antioxidant activities of these aque- (p < 0.05) than the control sample (0.72 ± ous solutions thus prepared without electrolysis or 0.04 nmole/ml, n = 4) (Table 2a). Therefore, TW extraction of a HPM-stick were also checked as in may act as a weak prooxidant in vitro on the human serum lipid. This will be discussed later. In the ex-periments of oxidation by oxygen in air of humanserum BR (2), the BR concentration in the freshly- RESULTS AND DISCUSSION
prepared reaction mixture for the control sample was1.15 ± 0.07 µg/ml (n = 4). At 17, 41 and 65 hr after The results of examinations for the inhibitory the preparation, the BR content in the control sample effects of tested aqueous solution systems on the decreased time–dependently to 0.95 ± 0.05 µg/ml oxidation of biological molecules are summarized (n = 4), 0.68 ± 0.06 µg/ml (n = 4) and 0.37 ± 0.05 µg/ in Table 2. In the experiments of oxidation by Cu2+ ml (n = 4), respectively. In the control sample, the of human serum lipid (1), the mean value and stan- quantities of BR, which disappeared due to oxida- dard deviation (S.D.) of the LPO concentration tion by oxygen in air, in the first 17 hr, the follow- in the control sample before the reaction was ing 24 hr and the last 24 hr, were 0.20 ± 0.02 µg/ml 0.97 nmole/ml and 0.05 nmole/ml (n = 4), respec- (n = 4), 0.27 ± 0.02 µg/ml (n = 4) and 0.31 ± 0.03 µg/ tively. After the reaction, it was elevated to be 1.69 ml, respectively. Such data obtained for all of sample ± 0.09 nmole/ml (mean ± S.D., n = 4), suggesting involving examined aqueous solution systems are that 0.72 ± 0.04 nmol/ml LPO (as MDA) was gen- summarized in Table 2b. As shown in Table 2b, un- erated by the action of 10 µM Cu2+. Mean values der the conditions employed, the BR quantities lost (± S.D.) for the LPO quantity generated in the by the oxidation in the first 17 hr in the samples in- samples involving ERW, IF, MSW, HT, TW, and volving ERW (0.09 ± 0.02 µg/ml, n = 4), IF (0.06 ± aqueous CFA with various volume ratios to DW in 0.01 µg/ml, n = 4), MSW (0.08 ± 0.01 µg/ml, n = 4), the reaction mixtures were calculated in the same HT (0.07 ± 0.01 µg/ml) and aqueous AsA (0.03 ± manner, respectively. All of such data are summa- 0.01 µg/ml, n = 4) were significantly lower rized in Table 2a. As shown in Table 2a, the LPO (p < 0.05) than the control sample (0.20 ± 0.02 µg/ quantities generated in the samples involving only ml, n = 4), respectively. It was therefore suggested ERW (0.59 ± 0.04 nmole/ml, n = 4), IF (0.53 ± that in the first 17 hr, oxidation by oxygen in air of 0.03 nmole/ml, MSW (0.56 ± 0.04 nmole/ml, n = 4), BR in the human serum was inhibited in vitro by HT (0.55 ± 0.04 nmole/ml, n = 4) and aqueous CFA ERW, IF, MSW and HT, although their inhibitory (0.18 ± 0.02 nmole/ml, n = 4) were significantly effects are considerably smaller than 100 µM aque- lower (p < 0.05) than that in the control sample (0.72 ous AsA. The antioxidant activities of all of these ± 0.04 nmole/ml, n = 4), respectively. Such effects aqueous solutions decreased time–dependently, and of the four aqueous solution systems and aqueous at 41 hr after the start of the reaction, the inhibitory CFA were reduced depending on decreases in their effects on the basis of significant differences in the volume ratios to DW in the reaction mixtures, al- values from those of the control sample were de- though the LPO quantities generated in the samples tected only in the sample involving aqueous AsA involving all of them with the volume ratios of 2 : 1 (Table 2b). The mechanisms of this phenomenon and the sample involving aqueous CFA with that of also will be discussed later. TW exhibited no sig- 1 : 2 were also significantly lower (p < 0.05) than nificant effect on the in vitro oxidation of human the control sample (Table 2a). These results suggest serum BR, although it tended to promote the oxida- that the generation of LPO by Cu2+ from human se- tion slightly (Table 2b). In the experiments of oxi- rum lipid is inhibited in vitro by the four aqueous dation of rutin by PPO (3), the rutin concentration solution systems examined which are assumed to in the control sample was 50.3 ± 2.2 µM (n = 4) and have antioxidant activities, although their inhibitory 13.5 ± 0.9 µM (n = 4) at the start and termination of effects are considerably smaller than 100 µM aque- the reaction, respectively. The quantity of rutin oxi- ous CFA. On the other hand, the LPO quantities gen- datively converted into the reaction products by the erated in the samples involving TW with various vol- action of PPO was 36.8 ± 1.9 µM (n = 4) in the con- Table 2. Effects of Sample Solutions on the Oxidation by Cu2+ of Human Serum Lipid (a), by Oxygen
on Air of Human Serum Bilirubin (b) and by Polyphenol Oxidase of Rutin (c) a: The Quantity of LPO (MDA) Generated (nmole/ml) (mean ± S.D., n = 4) a) Significantly greater or smaller (p < 0.05) than the control. Figures in the parenthesis indicate the values of MDA quantities generated during the corresponding periods.
b: The Content of Bilirubin (µg/ml) (mean ± S.D., n = 4) a) Significantly smaller (p < 0.05) than the control. Figures in the parenthesis indicate the bilirubin quantities oxidized during the corresponding periods.
c: The Quantity of Rutin Oxidized by the Enzyme (µM) (mean ± S.D., n = 4) a) Significantly smaller (p < 0.05) than the control. Abbreviations in the text.
trol sample. The quantities of rutin enzymatically shown in Table 2c, the rutin quantities oxidized in oxidized by PPO in the samples involving the four samples involving only ERW (31.3 ± 1.5 µM, n = 4), aqueous solution systems, aqueous AsA and TW with IF (27.6 ± 1.4 µM, n = 4), MSW (28.9 ± 1.9 µM, various volume ratios to DW were obtained in the n = 4), HT (30.6 ± 1.7 µM, n = 4) and aqueous AsA same manner. They are summarized in Table 2c. As (9.1 ± 0.8 µM, n = 4) were significantly smaller (p < 0.05) than the control sample (36.8 ± 1.9 µM, Table 3. Detection by ESR Spectrometry of the Scavenging
n = 4), respectively. These data suggest that the en- Ability for Oxygen Radicals of the Aqueous SolutionsExamined (Relative Values to Distilled Water for Man- zymatic oxidation of rutin is also suppressed by the four aqueous solution systems, although their effectsare not excellent as was observed for 100 µM aque- ous AsA. Such effects of these solutions were re- duced depending on increases in their volume ratios to DW in the reaction mixtures (Table 2c), as in the case of (1) (Table 2a). It has been known that PPO catalyzes the reaction for production of orthquinones from orthodiphenols including rutin and that the orthoquinones formed are then non-enzymatically polymerized to produce melanin-type pigments.9) Suppression by these aqueous solution systems of the oxidative transformation of rutin into the corre- Figures in the parenthesis indicate the % values of the inten- sponding orthoquinone(s) shows that they may act sity of signals for Oand ·OH against the distilled water sample.
as agents, which reduce (an) orthoquinone(s) to the a) and b) 6.58 and 12.37 as the values of intensity, respectively.
original substance. Effects of TW on the enzymatic Other abbreviations in the text. c) Electrolyzed reduced water, d) oxidation of rutin were not distinct (Table 2c).
”I’m Fine,” e) Mineral-stick water, f) Hira tenryosui water.
These results clearly suggest that all of the four examined aqueous solution systems, that are as-sumed to have antioxidant activities, really have such V3+, generally are reductive cations.10) It is therefore effects in vitro, that can be quantitated in compari- speculated that the antioxidant activities of IF and son with those of known antioxidants. No signifi- HT may be derived at least partly from such reduc- cant difference in the antioxidant activities detected tive V ions. This also will be discussed again later.
in the experiments for (1)–(3) were observed be- In MSW, Mg was the most abundant (Table 1).
tween samples of the four aqueous solution samples The results of examinations by ESR on the ROS examined, although IF tended to exhibit the stron- scavenging ability are presented in Table 3. As shown gest effects among them (Tables 2a–2c).
in Table 3, under the conditions employed, addition The results of the analyses of metal elements and of ERW, IF, MSW and HT instead of distilled water anions are summarized in Table 1. As shown in gave 21.8 (= 100.0 – 78.2)–76.2 (= 100.0 – 23.8)% Table 1, the concentrations of such minerals as iron reduction of signal intensity for O –, indicating that (Fe), copper (Cu), zinc (Zn), and strontium (Sr) and it was scavenged by the components of added solu- others were considerably higher in TW than in the tions. On the other hand, •OH was not scavenged at four aqueous solution systems. It is known that Fe all by ERW, MSW and HT or only slightly scav- and Cu can act as prooxidants based on the Fenton enged by IF (Table 3). It is therefore confirmed by reaction. The fact that TW slightly accelerated oxi- these ESR data that ROS scavengers, such as AsA, dation of serum lipid by Cu2+ (Table 2a) may be as- tocopherols, polyphenols and “active hydrogen” of sociated possibly with such properties of these met- which presence was assumed by Shirahata et al.,1) als. In ERW, Na was the most abundant cation, al- are virtually absent in these examined solutions, though a considerable amount of Cl– was also con- since such substances are excellent scavengers not tained (Table 1) presumably due to unblocked con- tamination of anions in electrolysis. In IF, MSW and A part of the results of experiments for exami- HT, the levels of all of examined anions were lower nation of the boiling and ultrafiltration treatments than in TW, while those of several metal elements on the antioxidant activities of the four aqueous so- were the prominently higher (Table 1). For instance, lution systems is summarized in Table 4. As show the concentrations of vanadium (V) and rubidium in Table 4, after boiling, inhibitory effects on the (Rb) and others in IF, as well as those of lithium Cu2+-induced oxidation of human serum lipid thor- (Li), V and Rb and others in HT, were far greater oughly disappeared in ERW and MSW, although in than TW (Table 1). It has been known that among V IF and HT, a part of the antioxidant activities con- ions, ones with the lower charge, such as V2+ and sistently remained. Similar trends were detected also Table 4. Effects of Boiling on the Inhibitory Effects of Aqueous
Solution Systems Examined on the Oxidation by Cu2+ ofHuman Serum Lipid Lipid peroxide generated (% to the values for DW)a) a) The data for the reaction mixtures involving samples and DW at the ratio of 3 : 0 (Reaction conditions in the text). Abbreviations in the text.
in the experiments of oxidation by oxygen in air of as above (elevation of the Mg level and no change human serum BR and of that by PPO of rutin (data in the antioxidant activities) were confirmed. The not shown). These data suggest that the antioxidant saturated concentration of hydrogen gas in an ERW activities of ERW and MSW are derived only from was reported to be 0.75 mM (= ca. 1.5 mg/l),12) which (a) volatile component(s) but those of IF and HT are should be accompanied by dissolution of ca. 18.2 mg generated also by (a) non-volatile component(s). The of metallic Mg into 1 l of DW. These results suggest ultrafiltration treatment gave virtually no effect on that hydrogen gas generated in association with dis- the antioxidant activities of any of these aqueous solution into the aqueous media of metallic Mg at solution systems, showing that the effective com- room temperature exhibits the antioxidant activities.
ponents are low MW substances which do not bind So, the HPM-stick of Water Institute, which seems to macromolecules or are not solid constituents with to consist mainly of metallic Mg, may be a product the larger pore size than that of the solutes with the so that the concentration of hydrogen gas dissolved is usually maintained within a narrow range near the An aqueous solution prepared by dissolving saturated state, compensating for the loss by evapo- NaCl and NaOH in TW to give the same Na and Cl– ration based on gradual extraction of metallic Mg concentrations as in ERW, as well as that prepared from the surface of the stick. It is not expected that by dissolving Mg(OH) in DW to afford the same hydrogen gas (molecular hydrogen) has a potent ability to scavenge ROS directly, although satura- METHODS), exhibited no antioxidant activity.
tion of the aqueous solution with hydrogen gas may The aqueous phase of Mg/DW at 1 hr after prepa- prevent dissolution of oxygen molecules, a part of ration of the reaction mixture (see MATERIALS These results suggest that hydrogen gas (molecu- same antioxidant activities as MSW in examinations lar hydrogen), which is generated by electrolysis (in for (1)–(3) (data not shown), and its Mg level by the ERW) and by dissolving metallic Mg as the major component of HPM-stick (in MSW), is an effective METHODS) was 16.3 mg/l, which was roughly component for the antioxidant activities of these equal to that by AAS of MSW (15.5 mg/l, Table 1).
aqueous solution systems. Thus, a part of what is The Mg concentration in the aqueous phase of the described in the recent HP of the Water Institute (the reaction mixture left at room temperature increased description that the MSW prepared according to the time–dependently to 23.1 mg/l at 24 hr later, while manual by users contains not “active hydrogen” but the antioxidant activities were not enhanced. The an- molecular hydrogen) [Water Institute (Atarashii- tioxidant activities in the aqueous phase of the reac- mizunokai); http://www.water-institute.org] has been tion mixture obtained at each time were time–de- confirmed to be right. This will be later discussed, pendently reduced after removal of metallic Mg by again. Molecular hydrogen as an effective compo- filtration (see MATERIALS AND METHODS), and nent for the antioxidant activities may be contained at 24 hr after the treatment, complete disappearance also in IF and HT. However, even its complete loss of the activities was found. On the other hand, when by boiling these solutions did not completely remove a HPM-stick was extracted by 1.5 ml of DW at room their antioxidant activities (Table 4). This may pos- temperature for the longer period, the same trends sibly be due to the presence of reductive V ion(s) as (a) solute(s) in these aqueous solution systems tive cation(s) dissolved as (a) solute(s). No phenom- (Table 1). The data that the antioxidant activities of enon which requires an assumption of the presence IF tended to be greatest among those of the four aque- of “active hydrogen,” occurs in association with the ous solution systems examined (Tables 2a–2c) may antioxidant activities of these aqueous solution sys- be given by the occurrence of both of generated molecular hydrogen and concentrated reductive V It has not yet been elucidated whether drinking ions during the process of electrolysis of the origi- these aqueous solution systems increases antioxidant nal natural water. Time–dependent reduction in the activities in human body fluids. This aspect remains antioxidant activities of the four aqueous solution to be investigated. However, even if molecular hy- systems (Table 2b) may be caused by evaporation drogen dissolved in these solutions is abundantly of dissolved hydrogen gas from their surface, and absorbed into the body of humans who drink them, also in IF and HT, by oxidation of V2+ and/or V3+ to it may be impossible that “active hydrogen” gener- V4+ and/or V5+ having no reductive effects.10) ated by the action of hydrogenase reacts with ROS These results indicate that generation of the an- in the human body, as recently described in the HP tioxidant activities in the four aqueous solution sys- of the Water Institute [Water Institute (Atarashii- tems examined can be explained solely by usual mizunokai); http://www.water-institute.org], because physico-chemical knowledge without assuming the humans have not this enzyme except that produced presence of “active hydrogen.” As described in the by some enterobacteria including Escherichia coli Introduction, Shirahata recently modified the “ac- (E. coli).16) So, even if a trace amount of “active tive hydrogen” hypothesis proposing that “active hydrogen” is formed in the intestine, it may be im- hydrogen” is accumulated on the surface of mediately consumed and can not be delivered to the microclusters of metals contained as nanocolloidal tissues where expression of its ROS scavenging abil- solid components in aqueous solution systems with antioxidant activities.5) A patent by Nihon Trim andShirahata for a method to prepare aqueous solution Acknowledgements
systems containing “active hydrogen”-accumulating thanks to Dr. Yuko Amo of Yamagata University for colloids,13) as well as that for a method to determine her helpful discussion. This study was aided by the “active hydrogen” in aqueous media,14) has been Grants of Japan Skeptics at 2002 and 2003. The pub- published. They also revealed the presence of Pt lication of this paper is supported by the Science nanoclusters, which was possibly released from the Research Frontier Program from the Ministry of electrodes during electrolysis, in an ERW they pre- Education, Sports, Science and Technology.
pared.15) In this study, as shown in Table 1, we de-tected only a trace amount (below 0.1 µg/l) of Pt asa solute in the ERW we prepared. This discrepancy REFERENCES
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grape seed extract and antioxidants by electron spin

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