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The reproducibility and reliability of the micro-β-carotene-linoleic acid bleaching (BCB) assay have been improved, enabling comparison to the antioxidant activity (AOA) of extracts from eleven kinds of crops evaluated by the oxygen radical absorbance capacity (ORAC) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) assays. All assays were conducted using 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) as a calibrator, and AOAs were expressed as micromoles of Trolox equivalent per gram of weight. The coefficient variation (CV) of the BCB assay using a microplate was 14.40%, which was comparable to that of the ORAC assay (H-ORAC (14.10%), L-ORAC (18.76 %)).Keywords: AOA, β-carotene bleaching assay, DPPH assay, ORAC assay, Trolox, plate reader, tocopherol *To whom correspondence should be addressed. E-mail: ichiho@affrc.go.jp IntroductionAntioxidants are believed to play an important role in preventing chronic illnesses such as heart disease, stroke, cancer, Alzheimer's disease, rheumatoid arthritis, and cataracts. As people have become more health-conscious, research on antioxidants has increased. Recently, reviews of AOA and measuring assays have been published (Koleva et al., 2002;Prior et al., 2005;Roginsky and Lissi 2004). These studies compared several commonly used assays for measuring AOA, and summarized the reaction mechanisms. Multiple reaction characteristics and mechanisms, as well as different phase localizations, were usually involved. Thus, no single assay accurately reflects all the radical sources or all the antioxidants in a system . In addition, antioxidants may respond differently to different radical or oxidant sources. Therefore, it is important at the outset to understand that AOA cannot be measured accurately and quantitatively by any simple universal assay .Antioxidants can deactivate radicals by two major mechanisms: hydrogen atom transfer (HAT), and single electron transfer (SET) (Huang et al., 2005). Therefore, we used the oxygen radical absorbance capacity (ORAC) assay (a HATbased method) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging assay (a SET-based method) to evaluate the AOA of crop extracts from Ibaraki Prefecture, Japan. We used the β-carotene bleaching (BCB) assay to evaluate the AOA of crop extracts and compared it to the AOA measured by the ORAC and DPPH assays. The BCB assay is based on the competitive bleaching of β-carotene during the antioxidation of linoleic acid in aqueous emulsion, and is monitored as decay of absorbance in the visible region (Miller, 1971). However, this assay is poorly quantified since the AOA is only given as an inhibition percentage and it is difficult to obtain repeatable data (Roginsky and Lissi, 2004). We therefore, for the first time, tried to express the AOA of the BCB assay as the Trolox equivalent. All three assays were conducted using the Trolox calibrator (positive control), and AOA was expressed as micromoles of the Trolox equivalent per gram of weight. In this study, we also ascertained the reproducibility and reliab...

Oxygen radical absorbance capacity (ORAC) value and polyphenol contents of extracts from 71 crops cultivated in Japan were determined. Hydrophilic ORAC values (H-ORAC) ranged from 194 − 29,830 μmol TE/100 g fresh weight (FW), whereas lipophilic ORAC values (L-ORAC) ranged from 6 − 2,076 μmol TE/100 g FW. High total ORAC value was indicated in the following vegetables and fruits: mulukhiya (Corchorus olitorius), perilla (Perilla frutescens), water spinach (Ipomoea aquatica), edible burdock (Arctium lappa), blueberries (Vaccinium corymbosum) and hardy kiwifruit (Actinidia arguta) (6,562 − 31,802 μmol TE/100 g FW). These extracts showed a strong positive correlation between ORAC value and polyphenol content (r = 0.98). In addition, to gain a better understanding of ORAC variability in Japanese crops, we also discussed the influence of environmental factors on the ORAC value of stem mustard (Brassica juncea) "Umino" grown in three different regions. Regional variations of ORAC in stem mustard likely result from different levels of ultraviolet radiation and fertilizer application regimens specific to those regions.

Further improvement of the thermostability of inherently thermostable proteins is an attractive challenge because more thermostable proteins are industrially more useful and serve as better scaffolds for protein engineering. To establish guidelines that can be applied for the rational design of hyperthermostable proteins, we compared the amino acid sequences of two ancestral nucleoside diphosphate kinases, Arc1 and Bac1, reconstructed in our previous study. Although Bac1 is a thermostable protein whose unfolding temperature is around 100°C, Arc1 is much more thermostable with an unfolding temperature of 114°C. However, only 12 out of 139 amino acids are different between the two sequences. In this study, one or a combination of amino acid(s) in Bac1 was/were substituted by a residue(s) found in Arc1 at the same position(s). The best mutant, which contained three amino acid substitutions (S108D, G116A and L120P substitutions), showed an unfolding temperature more than 10°C higher than that of Bac1. Furthermore, a combination of the other nine amino acid substitutions also led to improved thermostability of Bac1, although the effects of individual substitutions were small. Therefore, not only the sum of the contributions of individual amino acids, but also the synergistic effects of multiple amino acids are deeply involved in the stability of a hyperthermostable protein. Such insights will be helpful for future rational design of hyperthermostable proteins.

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