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Two potent antioxidative isoflavones were isolated from soybeans fermented with Aspergillus saitoi by silica gel column chromatography and preparative HPLC, using ODS column, or in addition, Toyopearl HW-40 column chromatography. The purified AS-13 and AS-9B compounds were identified as 8-hydroxydaidzein (8-OHD) and 8-hydroxygenistein (8-OHG), respectively, by MS, and (1)H-NMR, (13)C-NMR and HMBC spectra. These isoflavones, which have an o-dihydroxy structure between the 7- and 8-position, each exhibited significantly stronger antioxidative activity than daidzein and genistein in both oil and lipid/aqueous systems. Furthermore, the antioxidative activity and the content of each isoflavone analog in soybeans with different fermentation periods were investigated. It is suggested from these results that AS-13 and AS-9B were produced from daidzein and genistein, respectively, by hydroxylation at the 8-position of each isoflavone structure. In addition, it is concluded that these isoflavones were also the principal antioxidants in potent antioxidative soybeans fermented with A. saitoi.

The formation mechanism for the potent antioxidative o-dihydroxyisoflavones, 8-hydroxydaidzein (8-OHD) and 8-hydroxygenistein (8-OHG), was studied by incubating whole soybeans in a solid culture and a soybean extract in a liquid culture with Aspergillus saitoi. Analyses of changes in the isoflavone analogue content, β-glucosidase activity, and isoflavone hydroxylation ability indicated that 8-OHD and 8-OHG were formed from daidzein and genistein, respectively, by microbial hydroxylation, being respectively liberated from daidzin and genistin by β-glucosidase from A. saitoi during incubation. No selective hydroxylation reaction at the 8-position of daidzein and genistein were apparent during the vegetative stage, but were induced at the stage of sporulation.

Oxidative reactions during beer pasterization were studied using chemiluminescence (CL) and electron spin resonance (ESR) analyses. the CL production of beer was acclerated by pasteurization and the maximum CL intensity appeared sooner than that in non-pasteurized beer. Beers pasteurized with 15–30 pasteurization units (P.U) had the same CL producing activities as the non-pasteurized beers stored at 20°C for 6–10 days. Free radicals were produced and some of them were consumed by beer oxidation during pasteurization. It seems that free radicals were consumed by beer oxidation during pasteurization. It seems that free radical reactions occur in beer during pasteurization and that the degree of oxidation during pasteurization generally corresponded to that of non-pasteurized beer stored at room temperature for about 1 wee.

The effect of various isoflavones on the growth of human stomach cancer KATO III cells was investigated. Growth inhibition was greatest using genistein, followed by 8-hydroxygenistein (8-OHG), 8-hydroxydaidzein, and daidzein, respectively. Apoptosis induction in KATO III cells was observed following treatment with genistein, but not other compounds related to genistein or daidzein having the same isoflavone skeleton. Cells treated with genistein had morphological changes showing apoptotic bodies. The fragmentation of DNA by genistein to oligonucleosomal-sized fragments, a characteristic of apoptosis, was demonstrated to be concentration and time dependent by using agarose electrophoresis of DNA fragments isolated from the treated cells. The present study showed the positioning, but not the number of hydroxyl groups on the phenyl moiety of these isoflavones to be important for apoptosis-inducing ability.

One- and two-dimensional (1)H nuclear magnetic resonance ((1)H NMR) spectra were measured in order to estimate the dissociation constants (K(diss)) and molecular geometries of cyclodextrin (CD) with three organic isothiocyanates (ITC), allyl-ITC, 3-butenyl-ITC, and 4-pentenyl-ITC, in an aqueous solution (pH 5.0, I(c)=0.75 M). In every ITC, the K(diss) values decreased in the order of alpha-CDbeta-CDgamma-CD, and the three rod-like linear molecules of ITCs were accommodated with the best fit into the smallest cavity of alpha-CD. By rotating-frame nuclear Overhauser enhancement spectroscopy, cross peaks were found between the H-3 of alpha-CD and the H-1 of AITC. From the result, the molecular geometry of alpha-CD-AITC complex presume that the isothiocyanate group of AITC is located some where around the widening rim and a hydrophobic allyl group is included within the hydrophobic cavity of alpha-CD.

醤油醸造における産業廃棄物である醤油粕中のイソフラボン類の定量を行うとともに,この粕より各種抽出物,また8-OHGおよび8-OHDを高濃度に含有する画分を調製し,その抗酸化的有効利用の可能性を検討した.(1) 醤油もろみ中のイソフラボン類の粕への分配率は,約65∼95%を示した.ゲニステインの分配率が最も高く,これに次いで,ダイゼイン,8-OHG,8-OHDの順であった.(2) 各醸造メーカーの醤油粕中のイソフラボン類の総和量は,溜り醤油の粕の方が多かった.8-OHGおよび8-OHDの含量はメーカーによって異なっていたが,ODIの総和量は最高で100gあたり約60mgであった.(3) 醤油粕より調製したエタノール抽出物や酢酸エチル抽出物は,油系および水系における魚油の脂質過酸化を抑制した.8-OHGおよび8-OHDを高含量化したODI画分の抗酸化性はさらに強く,その活性はα-トコフェロール以上であった.(4) これらの醤油粕分画物は,DPPHラジカル捕捉能を示すとともに,リポソームに対しても強い抗酸化性を発揮した.特にODI画分の活性は顕著に強かった.以上の結果より,醤油粕より調製した分画物は種々の試験法で十分に抗酸化性を示すことが分かり,その有効利用の可能性が示唆された.またこの抗酸化性の発揮には,醤油粕中に多く残存する8-OHDや8-OHGが寄与するが,メラノイジンなどの各種抗酸化物質の関与も考えられる.

The effects of isothiocyanates (ITCs) on tumor necrosis factor-alpha (TNF-alpha) production by the J774A.1 mouse macrophage cell line stimulated with lipopolysaccharide (LPS) were examined. Some individual ITCs examined showed a priming effect, which was expressed as pre-activation, but also inhibited the production of TNF-alpha with concomitant stimulation by LPS, as a triggering stimulant of TNF production. These results suggest that ITCs exert opposing activities that can enhance or inhibit the host defense system.

醤油中の抗酸化成分を検索するとともに,各醸造メーカーより濃口醤油,溜り醤油および白醤油を入手し,これらに含まれる8-OHDおよび8-OHG含量を測定し,その抗酸化性を調べた.また,麹中のODI含量,ODIへの加熱の影響などを検討することにより,これらイソフラボン類の醤油中での抗酸化的役割を評価した.(1) 溜り醤油中の抗酸化成分を各種クロマトグラフィーにて分離し,8-OHDおよび8-OHGを主要な活性成分として単離・同定した.(2) これらのODIは,ダイズを主原料として製麹を行った各社の醤油麹に多く含有されていた.また,醸造を終えた醤油中にも抗酸化性を発揮する十分量のODIが存在した.溜り醤油中のODI含量は,仕込み時の加水量に大きく影響された.(3) コムギを主原料とした白醤油においては,麹中に含有されるODI量は少なかった.製品となる白醤油の中には,ODIが全く検出されないものもあった.(4) 白醤油に比較して,ODIが多く含まれる濃口醤油や溜り醤油の抗酸化力は強かった.特に,加水量の少ない6水溜りの活性は大きかった.ODI含有量と抗酸化活性との間には正の高い相関(r=0.959)が認められた.(5) 醤油中のODIは沸騰水中でこれを60分間加熱してもほとんど安定であり,その抗酸化性も低下しなかった.以上の結果より,8-OHDおよび8-OHGは,濃口醤油や溜り醤油中の主要な抗酸化成分であると考察される.また,これらのODIは醤油の醸造過程,さらには醤油を用いた各種加工食品においても,抗酸化的役割を果たしていると推察される.

豆味噌の製麹および熟成工程における8-OHD, 8-OHGおよび6-OHD含量の変動を測定した.またその抗酸化力等を調べることにより,これらODIの豆味噌製造時における抗酸化的役割を評価した.(1)原料ダイズ中のダイジンおよびゲニスチンは,味噌玉製麹中にβ-グルコシダーゼの作用により,それぞれダイゼインおよびゲニステインを生成した.これらの遊離イソフラボンは胞子形成期に生産される水酸化酵素の働きで8-OHD,6-OHDおよび8-OHGを生成した.これらのODI含量は製麹日数の経過とともに増加した.また,この麹の抗酸化力もODI含量の増加とともに増大した.(2)2日麹で仕込みを行った豆味噌中の8-OHG含量は,熟成の初期において増加した.その後の熟成期間中に僅かながらの減少傾向が認められたが,ほぼ一定の含量が保持された.8-OHDおよび6-OHD含量は約6ヶ月の熟成期間中,大きく変動することはなかった.(3)2日麹味噌の抗酸化力は熟成とともに増大した.この活性の増強は,特に8-OHG含量が増加した熟成初期において顕著であった.(4)4日麹味噌においても,ODI含量および抗酸化活性等の変動は2日麹味噌とほぼ同様であった.熟成を終えた4日麹味噌のODI含量はより高く,またその抗酸化性も2日麹味噌に比較して有意に高かった.以上の結果より,8-OHD,8-OHGおよび6-OHDは,豆味噌の製麹および長期にわたる熟成過程においても,脂質等の酸化的劣化の抑制に大いに貢献していると考察された.特に8-OHGは抗酸化力も強く,また味噌中での含量も高いことから,その抗酸化的役割は大きいと考えられる.これに次いで8-OHD, 6-OHDの順である.

The amino-carbonyl reaction (The Maillard reaction) of bovine lens crystallin, serum albumin or skin collagen with glucose was investigated to find effective means to prevent the formation of Advanced Glycation End Products (AGE) and induce the reversible solubilization of polymerized glycated proteins. The organic germanium compounds (Ge-132, 373, 385), derivatives of amino acids containing germanium as the linker of framework, were combined by the box titration method to determine the dose that would be most effective, compared with Aminoguanidine-HCl (AMG),α-tocopherol (VE), and pirenoxine (Catalin-K, CK). Although AMG suppressed the formation of AGE, effective concentrations were higher than 20 mM. Ge-385, when administered by itself at a low dose, induced the reversible solubilization of AGE made from crystallin, and albumin. The addition of any two reagents such as AMG, VE, CK and Ge-132 or 385 together to proteins lessened the effective range, and the peaks of smaller molecules in the profiles of HPLC and PAGE were quite remarkable. Examination was made of the effects of Ge-132 on the eyes of SAM mice, which show senescence accelerated cataracts at a relatively young age. The prevention of cataract-genesis and induction of reversible transparency of turbid lenses became evident following the administration of Ge-132 to the eyes 4 times a day. The mode of action of organic germanium compounds was demonstrated quite capable of disconnecting the sugar-parts from AGE by decarbonylation, resulting in the formation of glucosone and amino residues, and further leading subsequently to fewer AGE.

The decomposition of allyl isothiocyanate (AITC) in an aqueous solution was depressed in the presence of cyclodextrin (CD), it's suppression effect increasing in the order of nonebeta-CDalpha-CD. The results of kinetic and thermodynamic analysis of the CD-AITC inclusion complexes showed that the inclusion process was mostly governed by an enthalpy change (delta H degree) rather than by an entrophy change (delta S degree), and that Van der Waals forces played a primary role int he inclusion. Steric factors were important for the reaction activity of AITC inclusion into the CD cavity, especially significant being the stereospecificity between the size of the CD cavity and the AITC molecule which is the main factor concerning it's activity. Our results suggest that the association stability and activity of the included AITC molecule are important factors in the suppression mechanism for CDs. Therefore, both these factors would make an alpha-CD-AITC system more advantageous than a beta-CD-AITC system, and the marked suppression effect of alpha-CD on the decomposition of AITC can be attributed to the formation of inclusion complexes in an aqueous solution.

The effects of polyphenol oxidase (PPO) activity in wheat flour on the formation of protein-bound 5-S-cysteinyl-3, 4-di-hydroxyphenylalanine (5-S-cysteinyldopa) in gluten have been studied. The wheat was milled on a test mill. PPO activity of fractionated flour was determined by measuring the O2 consumption of aqueous suspension after addition of catechol, dopa or tyrosine as substrate. PPO activity of whole wheat toward tyrosine was lower than that toward catechol or dopa. Glutens prepared with whole wheat or short bran contain higher levels of protein-bound 5-S-cysteinyldopa than those prepared with white flour. PPO activity in wheat flour was related with formation amounts of protein-bound 5-S-cysteinyldopa in gluten. Heat treatment of whole wheat flour inhibited the formation of protein-bound 5-S-cysteinyldopa in gluten. When crude enzyme extracts prepared from wheat bran were added to dough, formation amounts of protein-bound 5-S-cysteinyldopa markedly increased. By the addition of enzyme-inhibitor, formation of protein-bound 5-S-cysteinyldopa in gluten was markedly decreased. It is believed that dough prepared with flour containing a high level of PPO activity has a network of protein-bound 5-S-cysteinyldopa formed from tyrosine and cysteinyl residue of protein.

(1) 水溶液中でのアリルイソチオシアナート(AITC)の分解に及ぼすアミノ酸の影響はアミノ酸の種類,その濃度により差異が認められた.影響の大きいアミノ酸としては疎水性グループでは,アラニン,シスチン,システィン,ロイシン,メチオニン,フェニルアラニン,トリプトファン,中性グループでは,スレオニン,チロシン,塩基性グループではアルギニン,リジンであった.これらアミノ酸は低濃度(1mM～4mM)の共存でもAITCの分解を促進した.(2) AITCとアミノ酸との相互反応はアミノ酸分子内に求核種のあるアミノ酸が大きく,AITCの分解を促進した.特に,システィンでは-SH基がその反応に関与していると考えられ,その反応性は他のアミノ酸の求核種に比べて極めて大いことが推測された.

The formation of protein-bound 5-S-cysteinyl-3,4-dihydroxyphenylalanine (5-S-cysteinyldopa) and 3,4-dihydroxyphenylalanine (dopa) as new cross-linkers in gluten was investigated. Gas-phase acid hyd...

To investigate the prevention of protein modification by food components, effects of natural antioxidants on in vitro modification of collagen by peroxynitrite were examined using a polyclonal antibody specific to 3-nitrotyrosine. To perform the assay with many samples at a time, the inhibitory effects of antioxidants were evaluated using an enzyme-linked immunosorbent assay. Polyphenols such as caffeic acid, curcumin, and flavonoids showed strong inhibitory effects on the formation of 3-nitrotyrosine in peroxynitrite-modified collagen. The inhibitory effects of caffeic acid and its related compounds on nitration of Tyr were then further investigated. Both caffeic acid, ferulic acid, and p-coumaric acid inhibited the modification, whereas cinnamic acid did not. By the treatment of p-coumaric acid with peroxynitrite, nitrated p-coumaric acid was detected in the reaction mixture using a liquid chromatograph-mass spectrometer (LC-MS). These results suggest that some phenolic antioxidants may prevent tissue i...

Steamed soybeans (SSB) were fermented with 30 kinds of Aspergillus strains which have been used in manufacturing Japanese fermented foods and beverages. Methanol extracts from the fermented soybean...

Many antioxidants have been found in spices and herbs, and some of them are well known as strong scavengers of active oxygen radicals. We have isolated active products, which markedly inhibited the formation of malondialdehyde (MDA from 2-deoxyribose and the hydroxylation of benzoate with the hydroxyl radical, from methanol extracts of allspice and clove. Pimentol from allspice, and biflorin and its isomer, abbreviated as clove3, from clove were identified as the active principles. These revealed strong activity as hydroxyl radical scavengers at a concentration of 2.0 microM. The antioxidative activities in an in vitro model system involving the rabbit erythrocyte membrane ghost were as strong as those of alpha-tocopherol at 200 microM. Such advanced glycation end products (AGE) as pentosidine are biomarkers of diabetes mellitus, and active oxygens have been suggested to be involved in the formation of AGE. The above-mentioned free radical scavengers effectively inhibited the formation of pentosidine in a model system of N alpha-t-butoxycarbonyl-fructoselysine and N alpha-t-butoxycarbonyl-arginine.

The effects of methanol extracts of 51 spices on ·OH scavenging were studied in detail. 2-Deoxyribose oxidation and sodium benzoic acid hydroxylation methods were used for detecting the scavenging activity of ·OH. Mustard varieties, thyme, oregano, clove, and allspice all exhibited strong ·OH-scavenging activity. In particular, 3 varieties of mustard had above 90% ·OH-scavenging activity with a 1 μg/ml concentration of their extracts. The ·OH scavenger of Brassica nigra (brown mustard) was isolated and purified by XAD-2 column chromatography and preparative HPLC, and was identified as a 3,5-dimethoxy-4-hydroxycinnamic acid methyl ester by MS, ‘H-NMR, and 13C-NMR. The 3,5-dimethoxy-4-hydroxycinnamic acid methyl ester was prepared by methylating of sinapic acid with diazomethane.

Recently, we have reported that anthocyanins show strong antioxidative activity, but no attention has been paid to anthocyanins from the viewpoint of the reaction mechanism of alkylperoxyl radicals; therefore, we investigated the reaction products of antioxidative anthocyanins (cyanidin 3-O-beta-D-glucoside). Cyanidin 3-O-beta-D-glucoside was reacted with 2,2'-azobis(2,4-dimet hylvaleronitrile) to generate the alkylperoxyl radicals, and the reaction products were isolated by high-performance liquid chromatography. The products were identified as 4,6-dihydroxy-2-O-beta-D-glucosyl-3-oxo-2,3-dihydroben-zofuran+ ++ and protocatechuic acid. Based on reaction products, the antioxidative mechanism of cyanidin 3-O-beta-D-glucoside may be different from that of alpha-tocopherol; cyanidin 3-O-beta-D-glucoside would produce another radical scavenger, as it would break down the structure and scavenge the radicals.

The behavior of sulfites in beer during fermentation and storage after packaging was studied using a high-performance liquid chromatographic-fluorescence detection method that was developed for det...

An analytical method was based on the aldehyde-bisulfite (separated by HPLC) reaction with o-phthalaldehyde in the presence of ammonium acetate, producing a fluorescent compound. Detection limits of alkanal-bisulfite (from formaldehyde- to octylaldehyde-bisulfite) and 2-alkenal-bisulfite (from trans-2-hexenal- to trans-2-nonenal-bisulfite) ranged from 5 nM to 25 nM. The method was less sensitive for free sulfite (detection limit 100 μM) than for aldehyde-bisulfite. Increasing acetaldehyde-bisulfite with addition of 25 mM acetaldehyde to a beer sample showed free sulfite in the beer. Free sulfite and acetaldehyde-bisulfite were detected in commercial beers but other aldehyde-bisulfites were not.

Dried tempeh is known to be significantly stable to lipid oxidation compared with unfermented soybeans (BS). A new antioxidant was isolated from the methanol extract of tempeh and identified as 3-hydroxyanthranilic acid (HAA) by UV, IR, EI-MS, and 1H-, 13C-, HMQC-, and HMBC-NMR techniques. HAA was effective in preventing autoxidation of soybean oil and soybean powder, while 6,7,4‘-trihydroxyisoflavone (a well-known antioxidant) was not. HAA also exhibited strong antioxidative activity in both water/ethanol and rabbit erythrocyte membrane ghost systems. HAA was not found in BS, but was produced during the incubations with Rhizopus oligosporus IFO 32002 and 32003. The amount of HAA reached a maximum at the stage of 2 days (acceptable), which had the strongest antioxidative activity. Keywords: Tempeh; fermented soybean product; antioxidant; 3-hydroxyanthranilic acid

Supercritical carbon dioxide (SC-CO 2 ) extraction was investigated for extraction of antioxidants from tamarind (Tamarindus indica L.) seed coat. Different combinations of pressure and temperature were used with and without ethanol as modifier. As temperature and pressure were increased, more antioxidants were extracted. Using ethanol as the modifier gave more extraction yield of antioxidant compounds. The SC-CO 2 extract with ethanol (30 MPa, 80 °C) was also a strong antioxidant for lard as well as linoleic acid autoxidation. These results suggest that the antioxidants included in tamarind seed coat can be extracted using SC-CO 2 and that they may be used for increasing the shelf life of foods by preventing lipid peroxidation.

We have been actively involved in the isolation and characterization of endogenous plant antioxidants that are believed to inhibit lipid peroxidation and offer protection against oxidative damage to membrane functions. Antioxidants have been isolated from conventional food sources, such as tea (green and black), sesame and wild rice, and also from other plant sources, such as rice hulls, and crude plant drugs. Data on new types of water-soluble and lipid-soluble plant antioxidants are provided, and the biological activity and functionality of these antioxidants are discussed.

In order to develop a new type of antioxidative compound which has both the phenolic and beta-diketone moiety in the same molecule, we converted three known curcuminoids, curcumin (diferuloylmethane, U1), (4-hydroxy-3-methoxycinnamoyl)methane (U2), and bis-(4-hydroxycinnamoyl)methane (U3), which are the natural antioxidants of Curcuma longa L. (tumeric), to tetrahydrocurcuminoids (THU1, THU2, and THU3, respectively) by hydrogenation, and evaluated their antioxidative activity by using linoleic acid as the substrate in an ethanol/water system. Further, we used the rabbit erythrocyte membrane ghost and rat liver microsome as in vitro systems and determined the antioxidative activity of these curcuminoids. When we evaluated their antioxidative activity by these assays, it was found that THU1 had the strongest antioxidative activity among all curcuminoids in each assay system. THU1 has been reported to be one of the main metabolites of U1 in vivo [Holder et al., Xenobiotica, 8, 761-768 (1978)]. These results suggest that THU1 must play an important role in the antioxidative mechanism of U1 in vivo by converting U1 into THU1.

タマリンド種皮抽出物の抗酸化性を食品加工へ利用するための基礎について検討した.(1) タマリンド種皮抽出物は,リノール酸モデル系において強い抗酸化性を示し,クエン酸及びα-トコフェロールとの間に相乗効果を示した.(2) タマリンド種皮抽出物は,100℃,2時間の加熱に対して安定であり,pHの変化に対しては,pH 5.0で抗酸化性の低下が認められた.また塩化ナトリウム存在下では,10%溶液中でも抗酸化性は,70%以上の残存率を示した.(3) 実際の食用油脂としてラード及びコーンサラダ油にタマリンド種皮抽出物を添加したところ,いずれの油脂においても抗酸化性を示した.

Effect various amino acids on the formation of 2, 3-dihydro-3, 5-dihydroxy-6-methyl-4 (H)-pyran-4-one (DDMP) and 5-hydroxymethylfurfural (HMF) have been investigated using a model system of cookie baking. A mixture of fructose and each amino acid was heated at 150°C for 10min in a test tube. Each reaction mixture was extracted with 1ml of water, and the extracts were analyzed by HPLC. DDMP was the major product in this reaction. The position of amino group affected on the formation of DDMP, and the amount of DDMP from fructose and β-amino acids was much more than that from fructose and other amino acids such as α- and γ-amino acids. The carbon chain length of α-amino acids also affected on the formation of DDMP, and DDMP was generated most remarkably in the reaction between fructose and α-alanine. When the carbon chain length was longer than that of α-alanine, DDMP formation decreased gradually. N-substituted arginine and lysine formed a large quantity of DDMP in the reaction with fructose than their N-unsubstituted amino acids. Basic amino acids reacted strongly and quickly with the sugar, and arginine also generated DDMP in the earlier stage of the reaction with fructose than β-alanine.

Antioxidative activity of tamarind (Tamarindus indica L.) seeds was investigated. An ethanol extract prepared from the seed coat exhibited antioxidative activity as measured by the thiocyanate and thiobarbituric acid (TBA) method, but there was no activity in the extract prepared from germ. The ethyl acetate extract prepared from seed coat had strong antioxidative activity. To determine antioxidative compounds in the seed coat chemically, preparative HPLC was carried out, then 2-hydroxy-3',4'-dihydroxyacetophenone (TAO), methyl 3,4-dihydroxybenzoate (TAI), 3,4-dihydroxyphenyl acetate (TA2), and (-)-epicatechin (TA3) were isolated from the ethyl acetate extract and identified

The antioxidative activity of the anthocyanin pigments cyanidin 3-O-β-D-glucoside (C3G) and cyanidin (Cy) was examined by using linoleic acid autoxidation, liposome, rabbit erythrocyte membrane, and rat liver microsomal systems. C3G and Cy had antioxidative activity in all systems. Cy had a stronger activity than C3G and the same activity as α-tocopherol (Toc) in the liposome and rabbit erythrocyte membrane systems. In the rat liver microsomal system, Cy and C3G exhibited stronger activity than Toc. These data suggest that the pigments may play an important role in the prevention of lipid peroxidation of cell membranes induced by active oxygen radicals in living systems as dietary antioxidants after ingestion

The mechanism of bisulfite adduct contribution to stability of beer quality was studied. Formaldehyde- and acetaldehyde-bisulffite adducts had only a poor oxidation potential around 0.93 V, which is the oxidation potential of free sulfite so that they lost their reducing activity for iodine. However, they inhibited the chemiluminescence production of beer and Cypridina lucifern analog dependent luminescence in beer, indicating that they inhibit the free radical reactions in beer. The acetaldehyde-bisulfite adduct inhibited haze formation and flavor staling of beer during storage. Therefore, it could be thought that aldehyde-bisulfite adducts have a radical scavenging activity and protect the free radical chain reactions during beer storage, leading to the stability of beer quality

The site-specific oxidation of histidine residues in glycated insulin mediated by copper ions and the relationship between the oxidation sites and the steric conformation of insulin are discussed in this study. Glycated insulin was prepared by incubating native insulin with glucose in 67 mM sodium phosphate, pH 7.5, at 37 degrees C for 30 h. In the presence of micromolar concentrations of Cu2+, glycated insulin was oxidized and its fragmentation or aggregation was detected. Accompanying the fragmentation, new N-termini were generated. The residues in these N-termini were identified as alanine, proline, valine, leucine and isoleucine by comparing dansyl derivatives with standard dansyl-amino acid products. Furthermore, several oxidized products of glycated insulin were isolated using reverse-phase HPLC (P1-P3). From amino acid composition and sequence analyses, it was determined that His10 on the insulin B-chain was modified in each of these peptides, while His5 was also modified in P3. The difference in susceptibility of His10 and His5 to oxidative modification is considered to be due to easier coordination of Cu2+ with His10, which further forms a complex with the Amadori compound at B-chain Phe1 that is vicinal to His10 in the steric conformation of insulin. This complex may generate an active oxygen species, which induces the degradation of the imidazole ring at His10, leading to aggregation or fragmentation of insulin.

The reactions of reducing sugars and amino acids were investigated by the heating of fructose or glucose with amino acids at 150 o C for 10 min as a model system for baking cookies. The products from the reaction were extracted with water and analyzed by HPLC. Fructose heated with β-alanine gave five nitrogen-containing compounds. One was identified as an Amadori compound (1-β-alanino-1-deoxyketose) by NMR and FAB-MS. The other three N compounds indicated the same molecular weight as that of the Amadori compound, i.e., m/z 251. The three N compounds are assumed to be 1-formyl-2-β-alaninoglucose, 1-formyl-2-β-alaninomannose, and 1-(hydroxymethyl)-2-β-alanino-2-deoxyketose

Riboflavin-sensitized photodynamic modification of collagen led to significant formation of cross-linked molecules. Sodium azide or 1,4-diazabicyclo(2,2,2)octane, which are known to be singlet oxygen quenchers, and catalase could not inhibit the modification. Surprisingly, the collagen modification was accelerated in the presence of superoxide dismutase. The aggregation was accompanied by the loss of tyrosine and histidine residues in the collagen. An inhibitory effect of dissolved oxygen on the modification of collagen was observed. Similarly, the loss of tyrosine residues in the irradiated collagen was inhibited in the presence of dissolved oxygen. Dityrosine formation was also observed with the loss of tyrosine. These results indicate that photodynamic modification of tyrosine probably contributes to the riboflavin-sensitized cross-linking of collagen through the formation of dityrosine.

Antioxidative activity of pea bean extract was examined. A TFA-ethanol extract prepared from red and black bean seed coat exhibited strong antioxidative activity. To determine the antioxidative role of the pigments in the seed coats, the pigments, RP0 (cyanidin 3-O-β-D-glucoside) and RP1 (pelargonidin 3-O-β-D-glucoside) from red bean and BP1 (delphinidin 3-O-β-D-glucoside) from black bean, were isolated. RPO showed strong antioxidative activity in the linoleic acid system at neutral condition (pH 7.0), while RP1 and BP1 exhibited no antioxidative activity at pH 7.0. In acidic conditions (pH 3.0 and 5.0), RP1 and BP1 showed strong antioxidative activity

Oxygenation of tryptophan analogues, 2-(3-indolyl)acetic acid (1), 3-(3-indolyl)propionic acid (2), N-benzoyl-L-tryptophan (3), n-tert-butoxycarbonyl-L-tryptophan (4), and W-benzoyl-L-tryptophan n-butylamide (5), with potassium superoxide (KO2)in dimethyl sulfoxide have been studied. In the case of 1, the major products were 2-formylaminobenzoic acid (25.3%) and 2′-formylaminoacetophenone (18.0%), which were produced by the secondary oxidation of 3-(2-formylaminophenyl)-3-oxopropinoic acid. The reaction of 2 with afforded 4-(2-formylaminophenyl)-4-oxobutanoic acid as the major product (43.3%), and 2-(3-dioxindolyl)acetic acid (14.5%) was also obtained. The reaction of 3 and 4 afforded 2-benzoylamino-4-(2-formylaminophenyI)-4-oxobutanoic acid (42.1%) and 2-tert-butoxycarbonylamino-4-(2-formylaminophenyl)-4-oxobutanoic acid (95.7%) as the major products, respectively. In the case of 3, 1 -benzoyl-2-carboxy-3a-hydroxy-1, 2, 3, 3a, 8, 8a-hexahydropyrrolo[2, 3-b]indole (27.4%) was also obtained. Unlike 3 and 4...

Methylglyoxal (MG), a precursor of advanced glycation end products, reacts rapidly with arginine residue in proteins. Three products, one fluorescent and two non-fluorescent adducts, have been so far identified. The fluorescent MG–arginine adduct, putatively identified as 5-methylimidazolone, has been shown to be identical to argpyrimidine. The non-fluorescent products were identified as the 5-hydro-5-methylimidazol-4-one and a novel MG adduct, which we named tetrahydropyrimidine (THP). THP was found to be formed from the reaction of guanidino group of arginine with two-MG molecules under physiological conditions. In the present study, to detect the THP adduct in vivo, we developed a monoclonal antibody (mAb5A3) directed to protein-bound THP. During preparation of the monoclonal antibody, hybridomas were selected by comparing the reactivities of the culture supernatant to THP using a competitive enzyme-linked immunosorbent assay. It was found that mAb5A3 specifically recognized THP as the dominant epitope. The presence of THP adduct was attested by immunohistochemical analysis of human atherosclerotic lesion, in which macrophage-derived foam cells were positively stained by the antibody. Our present results suggest that accumulation of THP occurs in vivo and may contribute to the progression of vascular diseases.

Antioxidative activity of pea bean (Phaseolus vulgaris L.) extract was evaluated by using a linoleic acid system, and the methanol extract exhibited strong antioxidative activity as measured by the thiocyanate method. The crude methanol extract was partitioned between then-butanol phase (BP) and the water phase (WP). Then, the antioxidative activity of the BP and the WP was determined by using a linoleic acid system. The WP showed strong antioxidative activity, while BP showed only weak activity as measured by the thiocyanate method. Next, the synergistic antioxidative action of WP with α-tocopherol was examined by using linoleic acid and liposome systems. The WP had a synergistic effect with α-tocopherol in both the food model and liposome systems. For purification and isolation of the antioxidative substances of the pea bean, preparative high-performance liquid chromatography was carried out with an octadecylsilyl column. Five fractions were collected, and antioxidative activity was determined in a linoleic acid system. Although fraction 1 had strong activity by the thiocyanate method, the purification of this active fraction was difficult; therefore, the partly characterized active fraction was investigated. The contents of total phenolics and sugars were 0.31±0.01 mg/g of fraction 1 and 406.1±0.1 mg/g, respectively. The ninhydrin chromogenic reaction was positive, and the ultraviolet absorption spectral λ max value in distilled water was 264.0 nm, indicating that the water-soluble antioxidative components from pea bean may be a new type of antioxidant. Isolation and identification are currently being investigated.

The oxidative damage of protein induced by the Amadori compound/copper(II) system was recently clarified (Kawakishi, S.; Okawa, Y.; Uchida, K.J. Agric. Food Chem. 1990, 38, 13-17). In the present study, we characterized a selective degradation and a possible cleavage mechanism for histidine residue found in protein mediated by the glycated peptide (Amadori compound)/copper(II) system. The main oxidized intermediate of the histidine residue, a 2-imidazolone derivative, was also confirmed in this system, which was similar to that formed in the ascorbate/copper(II) system. Although glucosone, a secondary product generated from copper(II)-catalyzed autoxidation of the Amadori compound, has been identified as a strong oxidizing agent causing protein damage, the Amadori compound itself was also confirmed to be an oxidizing agent similar to glucosone in the present study. A proposed mechanism for oxidative damage of protein mediated by autoxidation of the glycated peptide Amadori compound)/ copper(II) system is discussed.

PTCCからの黄色色素生成において, pH4.5ではPTCCは比較的安定であり,色素の生成も僅かであった.しかし, pHが上昇するに従い, PTCCが減少し,色素形成の増加が認められた.そして,両者間に高い負の相関が存在した.一方, McIlvaine緩衝液とリン酸緩衝液とには差は認められなかった.鉄(II)及び銅(II)により, PTCCの分解及び色素形成は10%程度増加した.一方, 5mMアスコルビン酸の添加により, pH4.5の区では, PTCCは安定であり,色素の生成も認められなかった.しかしpH5.5以上ではアスコルビン酸無添加区(Fig.1)と比較し,PTCCの分解および色素の形成は10%程度の減少であった.リン酸緩衝液(pH7.2)中で生成した黄色色素をセファデックスLH-20カラムで分離することにより,色素は変色し, PC上に新しく3個の蛍光物質が現れた.それらの化合物はいずれもβ-カルボリン骨格を有することが推定された.

The reactions of N -acyl-L-tryptophans, ( 1 ) and ( 2 , with O 2 − afforded their corresponding 2,3-bond cleavage compounds, ( 5 ) and ( 7 ), as the major products. On the other hand, the reactions of N -acyl-L-tryptophan alkylamides, ( 3 ) and ( 4 ), with O 2 − afforded their corresponding dioxindoles, ( 8 ) and ( 9 ), as the major products. A similar reactivity difference was observed for tryptophan-containing dipeptides, ( 10 ) and ( 11 ).

Glucosone has been identified as the main intermediate sugar moiety product of the copper(II)-catalysed autoxidation of the Amadori compound [Kawakishi, Tsunehiro & Uchida (1991) Carbohydr. Res. 211, 167-171]. Oxidative fragmentation of the model protein, especially selective degradation of the histidine residue in protein or peptides mediated by the copper(II)-catalysed autoxidation of glucosone, is discussed in this paper. The oxidative damage to protein could be retarded by catalase (EC 1.11.1.16) and EDTA, while superoxide dismutase (EC 1.15.1.1) and hydroxyradical scavengers showed little effect. Through the process of the oxidative degradation of N-benzoylhistidine and other histidine-containing peptides, the oxidation of the imidazole ring in histidine caused by the glucosone-copper(II) system was the same as that by the ascorbate-copper(II) system. These facts suggest that the copper-catalysed autoxidation of glucosone could generate some active-oxygen species causing oxidative damage to protein similar to that caused by the ascorbate-copper(II) system.

We found that glucosone (D-arabino-hexosulose), a typical enediol product formed in the Maillard reaction or in γ-radiolysis of sugars, was cytotoxic on Chinese hamster lung V79 cells in the presence of cupric ion (Cu 2+ ). Several other metal ions showed no effects. The cytotoxicity was assessed by colony formation assay and measurement of cell growth rate. In both assays, catalase inhibited the cytotoric effects. To confirm the role of H 2 O 2 on the cytotoxicity, we detected H 2 O 2 in the reaction mixture of glucosone and Cu 2+

The reactions of N -acyl-L-tryptophans, ( 1 ) and ( 2 , with O 2 − afforded their corresponding 2,3-bond cleavage compounds, ( 5 ) and ( 7 ), as the major products. On the other hand, the reactions of N -acyl-L-tryptophan alkylamides, ( 3 ) and ( 4 ), with O 2 − afforded their corresponding dioxindoles, ( 8 ) and ( 9 ), as the major products. A similar reactivity difference was observed for tryptophan-containing dipeptides, ( 10 ) and ( 11 ).