Masayoshi Sawamura

Spectrophotometric Assay for Superoxide Dismutase Based on the Reduction of Highly Water-soluble Tetrazolium Salts by Xanthine-Xanthine Oxidase

Two novel highly water-soluble tetrazolium salts, WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate sodium salt) and WST-8 (4-[3-(2-methoxy-4-nitrophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate sodium salt) were applied to the assay of superoxide dismutase (SOD). The superoxide anion generated by xanthine/xanthine oxidase (XO) reduced WST-1 and WST-8 to water-soluble formazans which exhibited absorbance maxima at 438 and 460 nm, respectively. The rates of reduction were linearly related to the XO activity, and reduction was inhibited by SOD. Complete inhibition by SOD of the reduction of both WST-1 and WST-8 was achieved, suggesting that these WSTs were not reduced with XO. WST-1 was found more useful than WST-8 because it had shown higher sensitivity which was apparently not dependent on the assay pH value in the range pH 8.0-10.2. These properties of WST-1 are ideal for the spectrophotometric assay of SOD in an aqueous system.

Characteristic Odor Components of Citrus reticulata Blanco (Ponkan) Cold-pressed Oil

Citrus reticulata Blanco (ponkan) cold-pressed oil and its oxygenated fraction were studied by analytical (GC and GC/MS) and sensory analyses. The monoterpene group was predominant, accounting for more than 89.6% (w/w), of which limonene was the most abundant (80.3%). Among the oxygenated compounds, octanal and decanal were the major ones among 12 aldehydes accounting for >1.5%; six alcohols were identified with a total concentration of >0.7%, while oxides, ketones and esters did not quantitatively or qualitatively contribute to the oil. Sniffing the ponkan cold-pressed oil and its oxygenated fraction demonstrated that octanal and decanal were the characteristic odor components of ponkan. Reconstruction of the ponkan aroma model and its sensory evaluation by a hedonic test were performed, showing that, in addition to octanal and decanal which played important roles, (R)-(+)-limonene contributed to the aroma model as a background component, making the aroma model very similar to that of the original.

Quantitative determination and characteristic flavour of Citrus junos (yuzu) peel oil.

Yuzu (Citrus junos Sieb. ex Tanaka) peel oils were extracted by cold-pressing. The flavour components of yuzu were quantitatively determined by use of two internal standards with GC and GC–MS. Characteristic flavour components of yuzu were examined by GC–olfactometry. The contents of hydrocarbons (34 compounds), alcohols (37 compounds) and aldehydes (16 compounds) in weight percent were about 96.55%, 2.03% and 0.1%, respectively. The terpenes were the major components of yuzu peel oil, but they contributed little to the characteristic flavour of yuzu. Methyltrisulfide, borneol, n-octanol, trans-undec-2-enal, (+)-p-mentha-1-en-9-ol, eugenol and carvacrol were found to be strongly involved in the characteristic flavour of yuzu, together with 6-methyl-5-hepten-2-ol and methyltrisulfide having the highest FD-factor. Copyright

Anti-inflammatory effects of limonene from yuzu (Citrus junos Tanaka) essential oil on eosinophils.

Yuzu (Citrus junos Tanaka) has been used as a traditional medicine in Japan. We investigated in vitro anti-inflammatory effects of limonene from yuzu peel on human eosinophilic leukemia HL-60 clone 15 cells. To examine anti-inflammatory effects of limonene on the cells, we measured the level of reactive oxygen species (ROS), monocyte chemoattractant protein-1 (MCP-1), nuclear factor (NF) kappa B, and p38 mitogen-activated protein kinase (MAPK). We found that low concentration of limonene (7.34 mmol/L) inhibited the production of ROS for eotaxin-stimulated HL-60 clone 15 cells. 14.68 mmol/L concentration of limonene diminished MCP-1 production via NF-kappa B activation comparable to the addition of the proteasomal inhibitor MG132. In addition, it inhibited cell chemotaxis in a p38 MAPK dependent manner similar to the adding of SB203580. These results suggest that limonene may have potential anti-inflammatory efficacy for the treatment of bronchial asthma by inhibiting cytokines, ROS production, and inactivating eosinophil migration.

Effect of Carnosine and Related Compounds on the Inactivation of Human Cu,Zn-Superoxide Dismutase by Modification of Fructose and Glycolaldehyde

Glycolaldehyde, an intermediate of the Maillard reaction, and fructose, which is mainly derived from the polyol pathway, rapidly inactivate human Cu,Zn-superoxide dismutase (SOD) at the physiological concentration. We employed this inactivation with these carbonyl compounds as a model glycation reaction to investigate whether carnosine and its related compounds could protect the enzyme from inactivation. Of eight derivatives examined, histidine, Gly-His, carnosine and Ala-His inhibited the inactivation of the enzyme by fructose (p<0.001), and Gly-His, Ala-His, anserine, carnosine, and homocarnosine exhibited a marked protective effect against the inactivation by glycolaldehyde (p<0.001). The carnosine-related compounds that showed this highly protective effect against the inactivation by glycolaldehyde had high reactivity with glycolaldehyde and high scavenging activity toward the hydroxyl radical as common properties. On the other hand, the carnosine-related compounds that had a protective effect against the inactivation by fructose showed significant hydroxyl radical-scavenging ability. These results indicate that carnosine and such related compounds as Gly-His and Ala-His are effective anti-glycating agents for human Cu,Zn-SOD and that the effectiveness is based not only on high reactivity with carbonyl compounds but also on hydroxyl radical scavenging activity.

Quantitative determination and characteristic flavour of daidai (Citrus aurantium L. var. cyathifera Y. Tanaka) peel oil

Daidai (Citrus aurantium L. var. cyathifera Y. Tanaka) peel oil was extracted by cold-pressing. The flavour components of daidai were quantitatively determined using two internal standards with GC and GC – MS. The characteristic flavour components of daidai were examined by GC – olfactometry. The percentages by weight of hydrocarbons (16 compounds), esters (12 compounds), alcohols (11 compounds) and aldehydes (10 compounds) were approximately 96.4%, 2.1%, 0.3% and 1.1%, respectively. Geranial, octyl acetate, geraniol, octanal, cedrol, cis-limonene oxide, trans-2-decenal, linalyl acetate and β-elemol were identified as characteristic flavour components of daidai. Terpenes were the major components of daidai peel oil, but they contributed little to the characteristic flavour of daidai. Linalyl acetate and β-elemol had the highest flavour dilution factors. The results of the omission test showed geraniol, octanal, linalyl acetate and cis-limonene oxide to be the active components of the daidai flavour. Copyright

Volatile components of peel cold-pressed oils of two cultivars of sweet orange (Citrus sinensis (L.) Osbeck) from Ethiopia

Cold-pressed fruit peel essential oils of two cultivars of sweet orange (Citrus sinensis (L.) Osbeck), Valencia and Hamlin, from Ethiopia were analyzed by capillary GC and GC–MS, without prior separation, and compared with each other. On two different GC columns, 58 components representing an average of 99.9% of the total volatile fraction were positively identified. Trans-carveol was proved for the first time as one of the components in cold-pressed orange oils. Monoterpene hydrocarbons followed by aldehydes and alcohols were the predominate quantitative composition of the volatile part in both the Valencia and Hamlin oils. Their percentage compositions, respectively, were 98.61% and 99.14% in total monoterpene hydrocarbons, 0.76% and 0.49% in aliphatic and terpenic aldehydes, and 0.47% and 0.18% in total alcohol concentration. In the cold-pressed oil of Valencia, oxygenated volatiles amounted to 1.26%, while in Hamlin amounted to only 0.70%. Compared to the same cultivars recently reported from other countries, high concentration of neral (0.09%) and geranial (0.16%) were found in Valencia orange oil. The major aliphatic aldehydes quantified in both oils are n-octanal and n-decanal, which are especially important character-impact constituents of sweet orange oils. Values for individual components like n-octanal, n-decanal and linalol were above, below or within values reported earlier. Copyright

Evaluation of Characteristic Aroma Compounds of Citrus natsudaidai Hayata (Natsudaidai) Cold-Pressed Peel Oil

The characteristic aroma compounds of Citrus natsudaidai Hayata essential oil were evaluated by a combination of instrumental and sensory methods. Sixty compounds were identified and quantified, accounting for 94.08% of the total peel oil constituents. Limonene was the most abundant compound (80.68%), followed by γ-terpinene (5.30%), myrcene (2.25%) and α-pinene (1.30%). Nineteen compounds which could not be identified in the original oil were identified in the oxygenated fraction. Myrcene, linalool, α-pinene, β-pinene, limonene, nonanal, γ-terpinene, germacrene D, and perillyl alcohol were the active aroma components (FD-factor > 36), whereas β-copaene, cis-sabinene hydrate and 1-octanol were suggested as characteristic aroma compounds, having a Natsudaidai-like aroma in the GC effluent. Three other compounds, heptyl acetate, (E)-limonene oxide and 2,3-butanediol, which each showed a high RFA value (>35) were considered to be important in the reconstruction of the original Natsudaidai oil from pure odor chemicals. The results indicate that 1-octanol was the aroma impact compound of C. natsudaidai Hayata peel oil.

Volatile Flavor Components of Ripe and Overripe Ki-mikans (Citrus flaviculpus Hort. ex Tanaka) in Comparison with Hyuganatsu (Citrus tamurana Hort. ex Tanaka)

The volatile flavor components of ripe and overripe ki-mikan (Citrus flaviculpus Hort. ex Tanaka) peel oil samples, which had been isolated by cold-pressing, were investigated by capillary GC and GC-MS, and compared with the Hyuganatsu (Citrus tamurana Hort. ex Tanaka) flavor. Limonene (ripe fruit, 82.44%; overripe fruit, 73.10%) was the most abundant compound in the ki-mikan oil, this being followed by γ-terpinene (8.83% and 13.74%), trans-β-farnesene (1.76% and 3.12%) and myrcene (1.54% and 1.13%). The composition of overripe ki-mikan oil was characterized by higher amounts of aliphatic and sesquiterpene hydrocarbons, monoterpene and sesquiterpene alcohols, ketones and esters than that of ripe ki-mikan oil. Monoterpene hydrocarbons, especially limonene (84.78%), were predominant in Hyuganatsu oil. The CPO composition of ki-mikan was qualitatively similar to that of Hyuganatsu, but differed quantitatively. The content of sequiterpene hydrocarbons was higher in the ki-mikan oil samples than in Hyuganatsu oil, while ketones showed the opposite predominance. These differences were more evident in the trans-β-farnesene and l-carvone contents. The ratio of both these compounds could be used to distinguish ki-mikan oil from Hyganatsu oil.

Chemical composition of agars from a newly reported Japanese agarophyte, Gracilariopsis lemaneiformis

The chemical composition of agars from Gracilariopsis lemaneiformis, newly reported from Japan, was investigated. Native agars were isolated by a sequential extraction of plants in water at 22 °C and 100 °C, and in boiling 20, 40 and 60° ethanol. Agars in each extract were analyzed by chemical methods, 1H, 13C NMR; and IR spectroscopy. The highest yield of agar (total carbohydrate) was obtained from the 40° ethanol extract (55°). Highest sulfate content was attained in non-alkali treated agars extracted with hot water (4.81°, DS 0.2). The 3,6-anhydrogalactose content was highest in the 40° ethanol extract (36.1° in non-alkali treatment, 40.3° in alkali treatment). The highest methoxyl content (6.51°, DS 0.66) was obtained in the 60° ethanol extract. The G. lemaneiformis agar is composed of the biological precursor to agarobiose repeating units and agarobiose containing 6-O-methyl agarobiose and a small amount of 2-O-methyl-α-l-galactopyranose residues. Alkali treatment improved the chemical quality of the agar fractions, which was comparable with Japanese commercial agar and agarose.