Jun Takebayashi

A method for evaluation of antioxidant activity based on inhibition of free radical-induced erythrocyte hemolysis.

There are many in vitro methods for evaluating antioxidant activity. In this chapter, we describe an operationally simple cell-based assay, oxidative hemolysis inhibition assay (OxHLIA). OxHLIA is based on inhibition of free radical-induced membrane damage in erythrocytes by antioxidants. The advantage of this method is that it uses peroxyl radicals as pro-oxidants and erythrocytes as oxidizable targets so that the results obtained reflect biologically relevant radical-scavenging activity and microlocalization of antioxidants. We also present here a comparison of OxHLIA with other common methods (DPPH, ABTS(*+), and ORAC assays).

Reassessment of antioxidant activity of arbutin: Multifaceted evaluation using five antioxidant assay systems

Abstract Arbutin, a practically used skin-lightening agent, has been reported to possess a weak antioxidant activity compared to that of its precursor, hydroquinone. However, its antioxidant activity has not been systematically evaluated. Hence, this study reassessed its activity using five assay systems. Assays were first performed using model radicals, DPPH radical and ABTS•+. Arbutin showed weak DPPH radical-scavenging activity compared to that of hydroquinone, but showed strong ABTS•+-scavenging activity. Its activity by ORAC assay was then evaluated using a physiologically relevant peroxyl radical. Arbutin exerted weak but long-lasting radical-scavenging activity and showed totally the same antioxidant activity as that of hydroquinone. Finally, it was shown that, in two cell-based antioxidant assays using erythrocytes and skin fibroblasts, arbutin exerted strong antioxidant activity comparable or even superior to that of hydroquinone. These findings indicate that the antioxidant activity of arbutin may have been under-estimated and suggest that it acts as a potent antioxidant in the skin.

Estimated Average Daily Intake of Antioxidants from Typical Vegetables Consumed in Japan : A Preliminary Study

The hydrophilic antioxidant content of 23 vegetables commonly consumed in Japan was assessed by the hydrophilic oxygen radical absorbance capacity (H-ORAC) method to estimate the dietary intake of total antioxidants in Japan. The estimated average H-ORAC value for “typical vegetables” consumed in Japan was 594.3 μmol Trolox equivalent (TE)/100 g. Hence, 2080 μmol TE/d of hydrophilic antioxidants would be ingested when 350 g of vegetables a day are consumed.

2-O-α-D-Glucopyranosyl-L-ascorbic Acid Scavenges 1,1-Diphenyl-2-picrylhydrazyl Radicals via a Covalent Adduct Formation

The 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging mechanism of 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G) was studied. We found two undefined products, named X and Y, in the reaction mixture of AA-2G and the DPPH radical under acidic conditions by HPLC analysis. The reaction mixture was further subjected to LC–MS analysis. X was found to be a covalent adduct of AA-2G and the DPPH radical. On the other hand, Y could not be identified, probably because it was a mixture. A time-course study of the radical-scavenging reaction revealed that one molecule of AA-2G scavenged one molecule of DPPH radical to generate an AA-2G radical, which readily reacted with another molecule of the DPPH radical to form a covalent adduct (X). Subsequently, this adduct slowly quenched a third molecule of the DPPH radical, resulting in reaction products (Y). Therefore, one molecule of AA-2G has only one oxidizable –OH group, but can scavenge three molecules of the DPPH radical. The radical-scavenging mechanism of AA-2G elucidated in this study should be useful in understanding the biological roles of AA-2G per se in the food and cosmetic fields.

Antioxidant Properties of 2-O-β- D -Glucopyranosyl- L -ascorbic Acid

The antioxidant activity of a provitamin C agent, 2-O-β-D-glucopyranosyl-L-ascorbic acid (AA-2βG), was compared to that of 2-O-α-D-glucopyranosyl-L-ascorbic acid (AA-2G) and ascorbic acid (AA) using four in vitro methods, 1,1-diphenyl-picrylhydrazyl (DPPH) radical-scavenging assay, 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS•+)-scavenging assay, oxygen radical absorbance capacity (ORAC) assay, and 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH)-induced erythrocyte hemolysis inhibition assay. AA-2βG slowly and continuously scavenged DPPH radicals and ABTS•+ in roughly the same reaction profiles as AA-2G, whereas AA quenched these radicals immediately. In the ORAC assay and the hemolysis inhibition assay, AA-2βG showed similar overall activities to AA-2G and to AA, although the reactivity of AA-2βG against the peroxyl radical generated in both assays was lower than that of AA-2G and AA. These data indicate that AA-2βG had roughly the same radical-scavenging properties as AA-2G, and a comprehensive in vitro antioxidant activity of AA-2βG appeared to be comparable not only to that of AA-2G but also to that of AA.

Determination of 15 isoflavone isomers in soy foods and supplements by high-performance liquid chromatography.

Soy isoflavone is the generic name for the isoflavones found in soy. We determined the concentrations of 15 soy isoflavone species, including 3 succinyl glucosides, in 22 soy foods and isoflavone supplements by high-performance liquid chromatography (HPLC). The total isoflavone contents in 14 soy foods and 8 supplements ranged from 45 to 735 μg/g and from 1,304 to 90,224 μg/g, respectively. Higher amounts of succinyl glucosides were detected in natto, a typical fermented soy product in Japan; these ranged from 30 to 80 μg/g and comprised 4.1-10.9% of the total isoflavone content. In soy powder, 59 μg/g of succinyl glucosides were detected, equivalent to 4.6% of the total isoflavone content. These data suggest that the total isoflavone contents may be underestimated in the previous studies that have not included succinyl glucosides, especially for Bacillus subtilis -fermented soy food products.

Extraction Efficiency of Hydrophilic and Lipophilic Antioxidants from Lyophilized Foods Using Pressurized Liquid Extraction and Manual Extraction

The efficient extraction of antioxidants from food samples is necessary in order to accurately measure their antioxidant capacities. α-Tocopherol and gallic acid were spiked into samples of 5 lyophilized and pulverized vegetables and fruits (onion, cabbage, Satsuma mandarin orange, pumpkin, and spinach). The lipophilic and hydrophilic antioxidants in the samples were sequentially extracted with a mixed solvent of n-hexane and dichloromethane, and then with acetic acid-acidified aqueous methanol. Duplicate samples were extracted: one set was extracted using an automated pressurized liquid extraction apparatus, and the other set was extracted manually. Spiked α-tocopherol and gallic acid were recovered almost quantitatively in the extracted lipophilic and hydrophilic fractions, respectively, especially when pressurized liquid extraction was used. The expected increase in lipophilic oxygen radical absorbance capacity (L-ORAC) due to spiking with α-tocopherol, and the expected increase in 2,2-diphenyl-1-picrylhydrazyl radical scavenging activities and total polyphenol content due to spiking with gallic acid, were all recovered in high yield. Relatively low recoveries, as reflected in the hydrophilic ORAC (H-ORAC) value, were obtained following spiking with gallic acid, suggesting an interaction between gallic acid and endogenous antioxidants. The H-ORAC values of gallic acid-spiked samples were almost the same as those of postadded (spiked) samples. These results clearly indicate that lipophilic and hydrophilic antioxidants are effectively extracted from lyophilized food, especially when pressurized liquid extraction is used.

Improvement of the Lipophilic-Oxygen Radical Absorbance Capacity (L-ORAC) Method and Single-Laboratory Validation

We improved the procedure for lipophilic-oxygen radical absorbance capacity (L-ORAC) measurement for better repeatability and intermediate precision. A sealing film was placed on the assay plate, and glass vials and microdispensers equipped with glass capillaries were used. The antioxidant capacities of food extracts can be evaluated by this method with nearly the same precision as antioxidant solutions.

Effect of docosahexaenoic acid intake on lipid peroxidation in diabetic rat retina under oxidative stress

Docosahexaenoic acid (DHA) plays an important role in visual function but has a highly oxidation-prone chemical structure. Therefore, we investigated how dietary DHA affects the generation of lipid peroxides in rat retina under oxidative stress in diabetes with/without vitamin E (VE) deficiency. Streptozotocin-induced (50 mg i.p./kg B.W.) diabetic Sprague–Dawley (SD) rats were assigned to four groups: (i) control/VE(+), (ii) DHA/VE(+), (iii) control/VE( − ) and (iv) DHA/VE( − ), and raised for 28 days. We then measured lipid peroxide levels in the retina, serum and liver. With a normal intake of VE, dietary DHA increased only the retinal level of thiobarbituric acid-reactive substances (TBARS) slightly. In contrast, in rats with VE deficiency, dietary DHA increased serum and liver lipid peroxide levels but not in the retina. These results suggest that dietary DHA does not necessarily promote lipid peroxidation in the retina even under high oxidative stress.

Improvement and Interlaboratory Validation of the Lipophilic Oxygen Radical Absorbance Capacity: Determination of Antioxidant Capacities of Lipophilic Antioxidant Solutions and Food Extracts.

A lipophilic oxygen radical absorbance capacity (L-ORAC) assay is an evaluation of the antioxidant capacity of solutions of lipophilic compounds. The concentrations of fluorescein, radical generator, and Trolox standard solutions were optimized to improve the precision of the assay. An interlaboratory study using two antioxidant solutions and three food extracts as test samples conducted in accordance with harmonized protocol demonstrated satisfactory L-ORAC measurements; the intermediate precision relative standard deviations (RSD(int)) ranged from 7.0 to 16.7%, the reproducibility relative standard deviations (RSD(R)) ranged from 14.8 to 19.4%, and the HorRat values ranged from 1.35 to 1.78.