Kinetics for Singlet Oxygen Formation by Riboflavin Photosensitization and the Reaction between Riboflavin and Singlet Oxygen

ABSTRACT: The formation of singlet oxygen by riboflavin and the kinetics and mechanisms of riboflavin degradation in aqueous solution under light were determined. The singlet oxygen formation rate by riboflavin was 2.31 μmole oxygen/mL headspace/h of serum bottle. The degradations of riboflavin were 66% in D₂O and 40% in H₂O, respectively, under light after 24 h. The results indicate that singlet oxygen is involved in riboflavin destruction under light. The riboflavin destructions were 94.0% and 15.7% with 0 mM or 160 mM ascorbic acid, respectively, under light after 96 h. The reaction rate between riboflavin and singlet oxygen was 1.01 × 10¹⁰/M/s, which is a diffusion-controlled reaction rate. This explains the extremely fast degradation of riboflavin in foods under light. Ascorbic acid and sodium azide reduce the degradation of riboflavin under light with different quenching mechanisms. Ascorbic acid quenched both singlet oxygen and excited triplet riboflavin. Sodium azide quenched only the singlet oxygen in riboflavin solution with a quenching rate of 1.547 × 10⁷/M/s. With the involvement of both the Type-I and Type-II mechanisms in the riboflavin degradation under light, singlet oxygen quencher alone could not protect the riboflavin from degradation completely. Addition of ascorbic acid can protect riboflavin oxidation in foods exposed to light.     

Riboflavin Photosensitized Singlet Oxygen Oxidation of Vitamin D

Samples containing various levels of vitamin D and riboflavin, with and without ascorbic acid or a-tocopherol were prepared in a model system. Samples were stored in the light or in the dark at 45®C for up to 8 h. Headspace oxygen was determined by gas chromatography with thermal conductivity detection. Oxidation of vitamin D was not observed in samples without riboflavin stored in the light nor in samples with riboflavin stored in the dark. Vitamin D with riboflavin was oxidized under light. α-Tocopherol quenched singlet oxygen at a rate of 2.52 × 10⁸ M^-1 sec^-1, whereas ascorbic acid quenched singlet oxygen at a rate of 2.23 × 10⁷ M^-1 sec^-1.     

Singlet Oxygen Quenching Effects of Phosphatidylcholine in Emulsion Containing Sunflower Oil

ABSTRACT:  Effects of phosphatidylcholine (PC) on the oxidation of oil by singlet oxygen in a W/O microemulsion and an emulsion food model containing tocopherol-stripped sunflower oil (TSSO) have been studied. The W/O microemulsion consisted of methylene chloride, butanol, and sodium dodecyl sulfate with PC (0, 250, 1000 ppm) and TSSO (0, 3.3, 16.5, 33 mg/mL). Production of singlet oxygen in the microemulsion was done chemically with hydrogen peroxide in the presence of sodium molybdate, and indirectly evaluated by rubrene oxidation at A₅₂₉. The emulsion food model consisted of TSSO, distilled water, and xanthan gum with addition of 250 ppm PC and 4 ppm chlorophyll b, and was placed at 25 °C under fluorescent lights (1700 lux) for 24 h. The oxidation of TSSO was determined by thin-layer chromatography and values of conjugated dienoic acid (CDA) and peroxides (POV). PC significantly decreased the oxidation of rubrene and TSSO in the W/O microemulsion, but its content was decreased to approximately one-half by a 20-min reaction, indicating its degradation. This clearly shows that PC acted as an antioxidant via chemical quenching of singlet oxygen in the W/O microemulsion. A possible synergism between PC and TSSO was observed in singlet oxygen quenching in the microemulsion. PC also significantly decreased the chlorophyll-photosensitized oxidation of TSSO in the emulsion food model, possibly by singlet oxygen quenching. This study clearly suggested that PC be used as an antioxidant to improve the lipid oxidative stability of an emulsion food containing chlorophyll under light.     

Direct Spectroscopic Observation of Singlet Oxygen Quenching and Kinetic Studies of Physical and Chemical Singlet Oxygen Quenching Rate Constants of Synthetic Antioxidants (BHA,BHT, and TBHQ) in Methanol

Abstract: Singlet oxygen quenching by synthetic antioxidants (BHA, BHT, and TBHQ) was directly observed by spectroscopic monitoring of luminescence at 1268 nm. The luminescence data showed unambiguous evidence of singlet oxygen quenching by synthetic phenolic antioxidants with the highest activity for TBHQ, followed by BHA and BHT. The protective activities of these synthetic antioxidants on α-terpinene oxidation with chemically-induced singlet oxygen under dark further confirmed their singlet oxygen quenching abilities. Total singlet oxygen quenching rate constants (k_r + k_q) of BHA, BHT, and TBHQ were determined in a system containing α-terpinene (as a singlet oxygen trap) and methylene blue (as a sensitizer) during light irradiation, and the values were 5.14 × 10⁷, 3.41 × 10⁶, and 1.99 × 10⁸ M⁻¹s⁻¹, respectively. After the k_r value of α-terpinene was first determined, the k_r values of the synthetic antioxidants were calculated by measuring their relative reaction rates with singlet oxygen to that of α-terpinene under the identical conditions. The k_r values of the BHA, BHT, and TBHQ were 3.90 × 10⁵, 1.23 × 10⁵, and 2.93 × 10⁶, M⁻¹s⁻¹. The percent partition of chemical quenching over total singlet oxygen quenching (k_r × 100)/(k_r + k_q) for BHA, BHT, and TBHQ were 0.76%, 3.61%, and 1.47%, respectively. The results showed that the synthetic antioxidants quench singlet oxygen almost exclusively through the mechanism of physical quenching. This represents the first report on the singlet oxygen quenching mechanism of these synthetic antioxidants. Practical Application: The synthetic antioxidants, especially TBHQ, have been found to have a strong singlet oxygen quenching ability. This article also clearly showed that singlet oxygen quenching by synthetic antioxidants was mainly by the physical quenching mechanism. The results suggested that these synthetic antioxidants, especially TBHQ, could be used practically for the protection of the food components such as edible oils and vitamins against singlet oxygen induced oxidations without significant losses of antioxidant activity during storage under light.     

Effects of Riboflavin Photosensitized Oxidation on the Volatile Compounds of Soymilk

ABSTRACT: Soymilks with or without added riboflavin in serum bottles were stored under light or in dark at 20 °C. The headspace oxygen and volatile compounds were determined by gas chromatography. Riboflavin had significant effects on the headspace oxygen depletion and volatile compounds formation in soymilk under light ( P < 0.05). Riboflavin did not have significant effects on the formation of volatile compounds and the depletion of headspace oxygen in dark ( P > 0.05). The volatile compounds increased under light, but not in dark as the added riboflavin increased. Storage temperature at 4 °C or 20 °C did not have significant difference in the effect of riboflavin on the headspace oxygen depletion in soymilk under light. Hexanal, an important beany flavor compound, was identified as the major volatile compound in the riboflavin photosensitized soymilk. Singlet oxygen oxidation was involved in the formation of volatile compounds in soymilk under light. Hexanal could be formed by singlet oxygen oxidation. Ascorbic acid, a quencher for singlet oxygen and the excited triplet sensitizer, significantly inhibited the formation of hexanal and total volatiles in soymilk under light.     

Effects of Riboflavin Photosensitization on the Degradation of Bisphenol A (BPA) in Model and Real-Food Systems

ABSTRACT:  Effects of riboflavin photosensitizations on the stability of bisphenol A (BPA), a well-known endocrine disrupting chemical, were studied in model and real-food systems by high-performance liquid chromatography (HPLC). Concentration of BPA was significantly decreased under light exposure ( P < 0.05) as the concentration of riboflavin increased while those without riboflavin under light or those with riboflavin in the dark did not change significantly ( P > 0.05). Addition of 50, 100, and 200 μM sodium azide significantly increased the stability of BPA in riboflavin photosensitization with concentration dependent manner ( P < 0.05), implying that a singlet oxygen or type II pathway played a role in the photodegradation of BPA. Stability of BPA in riboflavin was significantly increased in the presence of high concentration of tert -butanol, a hydroxyl radical quencher, under light storage for 80 min, indicating hydroxyl radicals were involved and contributed to the degradation of BPA, at least in part. Availability of riboflavin photosensitization on the photodegradation of BPA was tested in 2 canned tea beverages with different phenolic contents. BPA was more stable in the beverage sample with higher total phenolic contents and free radical scavenging ability. The photodegradation of BPA in riboflavin photosensitization can be an efficient way to decrease the concentration of BPA from environmental or food systems.     

Kinetic Study of the Quenching Reaction of Singlet Oxygen by Common Synthetic Antioxidants (tert-Butylhydroxyanisol, tert-di-Butylhydroxytoluene, and tert-Butylhydroquinone) as Compared with α-Tocopherol

ABSTRACT: Effects of synthetic phenolic antioxidants (BHA, BHT, and TBHQ) on the methylene blue (MB) sensitized photooxidation of linoleic acid as compared with that of α‐tocopherol have been studied. Their antioxidative mechanism was studied by both ESR spectroscopy in a 2,2,6,6‐tetramethylpiperidone (TMPD)‐methylene blue (MB) system and spectroscopic analysis of rubrene oxidation induced by a chemical source of singlet oxygen. Total singlet oxygen quenching rate constants (k_ox?Q+k_q) were determined using a steady state kinetic equation. TBHQ showed the strongest protective activity against the MB sensitized photooxidation of linoleic acid, followed by BHA and BHT. TBHQ (1 × 10^?3 M) exhibited 86.5% and 71.4% inhibition of peroxide and conjugated diene formations, respectively, in linoleic acid photooxidation after 60‐min light illumination. The protective activity of TBHQ against the photosensitized oxidation of linoleic acid was almost comparable to that of α‐tocopherol. The data obtained from ESR and rubrene oxidation studies clearly showed the strong singlet oxygen quenching ability of TBHQ. The k_ox?Q+k_q of BHA, BHT, and TBHQ were determined to be 3.37 × 10⁷, 4.26 × 10⁶, and 1.67 × 10⁸ M^?1 s^?1, respectively. The k_ox?Q+k_q of TBHQ was within the same order of magnitude of that of α‐tocopherol, a known efficient singlet oxygen quencher. There was a high negative correlation (r² = ?0.991) between log (k_ox?Q+k_q) and reported oxidation potentials for the synthetic antioxidants, indicating their charge‐transfer mechanism for singlet oxygen quenching. This is the 1st report on the kinetic study on k_ox?Q+k_q of TBHQ in methanol as compared with other commonly used commercial synthetic antioxidants and α‐tocopherol.     

食品中脂类物质的1O2氧化及控制方法

脂类食品中存在的天然色素通常是潜在的光敏剂,可以通过光敏反应Type II途径将基态氧激发并转变为单线态氧（singlet oxygen,1O2）。1O2是一种亲电性、非自由基的激发态氧分子,具有活化能低、反应活性高的特点,可以快速引发脂类物质氧化,并最终导致其氧化劣变。本文查阅并分析了国内外本领域的最新研究成果,对脂类食品中1O2的形成途径、光敏氧化机理及甘油三酯和植物甾醇等常见脂类物质的光敏氧化进行了深入阐述,并从光敏剂和三线态氧的脱除与控制、1O2淬灭、光源选择等方面总结了食品中脂类物质光敏氧化控制的最新研究进展,以期为深入研究食品中脂类物质的光敏氧化及抑制技术提供理论依据。     

Singlet Oxygen and Ascorbic Acid Effects on Dimethyl Disulfide and Off-Flavor in Skim Milk Exposed to Light

The light-induced off-flavor and dimethyl disulfide increased as fluorescent light exposure increased from 0,2,4,6, to 8h (p<0.05). Sensory evaluation and identified compounds suggest that dimethyl disulfide was mainly responsible for the light-induced off-flavor in skim milk. Dimethyl disulfide was formed by the singlet oxygen oxidation of methionine in milk. Gas chromatographic analysis and sensory evaluation showed that 200,500, and 1000 ppm ascorbic acid, which is a singlet oxygen quencher, lowered the formation of dimethyl disulfide and off-flavor in skim milk (p<0.05).     

Singlet Oxygen Quenching Activities of Various Fruit and Vegetable Juices and Protective Effects of Apple and Pear Juices against Hematolysis and Protein Oxidation Induced by Methylene Blue Photosensitization

ABSTRACT:  Effects of various fruit and vegetable juices on rubrene oxidation induced by a chemical source of singlet oxygen in a microemulsion system have been studied. The singlet oxygen quenching activities of fruit and vegetable juices were greatly different with different juices. The apple and pear juices exhibited the highest antioxidative activity among the tested juices in singlet oxygen–induced rubrene oxidation, showing 56.69% and 59.34% inhibition, respectively. The grape, kumquat, red cabbage, and spinach juices also showed relatively strong antioxidative activity against singlet oxygen–induced rubrene oxidation. Lemon juice showed the least activity, resulting in 0.63% inhibition of rubrene oxidation. The singlet oxygen quenching activities of 1 mL of apple and pear juices were equivalent to 33.97 and 34.64 mg ascorbate, respectively. Singlet oxygen quenching activities of juices had very low correlation with both ABTS radical scavenging activity ( R ²= 0.11) and total phenolic contents ( R ² < 0.1). However, a high correlation ( R ²= 0.66) was found between the ABTS radical scavenging activities and total phenolic contents of juices. The apple and pear juices also significantly inhibited both erythrocyte lysis and protein oxidation induced by fluorescence light illumination in the presence of methylene blue. Electron spin resonance (ESR) spectroscopy data showed that the protective activities of these juices against biological damages induced by photodynamic ways were, to at least some extent, due to their singlet oxygen quenching abilities. This represents first report on the singlet oxygen quenching activities of the apple and pear juices, and their protective activities against photodynamically induced biological damages.     

Antioxidant mechanisms of Trolox and ascorbic acid on the oxidation of riboflavin in milk under light

Antioxidant activities and the mechanism of water-soluble Trolox and ascorbic acid on the oxidation of riboflavin in milk were studied. Trolox or ascorbic acid at 0, 100, 250, 500, or 1000 ppm was added to milk with or without added 50 ppm riboflavin and stored under light at 27 °C. Headspace oxygen was analysed by GC and Trolox, ascorbic acid, and riboflavin were determined by HPLC. The headspace oxygen of milk with added 50 ppm riboflavin depleted faster than that of milk without added riboflavin (p < 0.05). Trolox and ascorbic acid decreased during storage under light and riboflavin was completely destroyed within 24 h. As the concentration of Trolox or ascorbic acid increased, the riboflavin loss decreased. Riboflavin, Trolox, and ascorbic acid competed to react with singlet oxygen which was formed in the presence of riboflavin under light. Trolox and ascorbic acid protected riboflavin in milk under light by reacting with singlet oxygen.     

Effects of Trolox and ascorbic acid on the riboflavin photosensitised oxidation of aromatic amino acids

The effects of 0, 100, 250, 500, 750 or 1000 ppm Trolox and ascorbic acid on the singlet oxygen oxidation of aromatic amino acids viz. phenylalanine, tryptophan, tyrosine in the presence of 25 ppm riboflavin were determined by measuring depleted headspace oxygen by gas chromatography and aromatic amino acid content by high performance liquid chromatography. The coefficients of variations (CVs) for headspace oxygen analysis and HPLC analysis of aromatic amino acids were 1.4% and 4.7%, respectively. Samples were stored under light (1000 lux) at 30 °C for 10 h. Both Trolox and ascorbic acid acted as antioxidants. As the concentration of Trolox and ascorbic increased from 0 to 1000 ppm, the head space oxygen depletion increased. This was due to the oxidation of Trolox and ascorbic acid along with amino acids in the presence of riboflavin. High performance liquid chromatography analysis of the samples clearly indicated that both Trolox and ascorbic acid minimised the degradation of phenylalanine, tryptophan, tyrosine significantly (p < 0.05), but did not prevent their oxidation completely. Trolox acted as a better antioxidant than ascorbic acid in protecting phenylalanine, tryptophan and tyrosine. Type-I mechanism was mainly responsible for riboflavin photosensitised degradation of aromatic amino acids.     

游离态叶黄素和玉米黄素体外单线态氧淬灭率的比较研究

为比较玉米黄素和叶黄素单体的体外淬灭单线态氧能力,本实验分别萃取万寿菊与枸杞中的叶黄素与玉米黄素酯,经皂化和纯化后得到游离的叶黄素与玉米黄素组分。使用C30-HPLC-PDA 分别对制备组分进行定性和定量分析。用次氯酸钠-双氧水反应产生单线态氧,使用微弱发光测量仪测定制备组分对单线态氧的淬灭率。结果显示：游离叶黄素和玉米黄素均能减少次氯酸钠-双氧水反应体系产生的单线态氧数量,导致最大光子计数值下降。二者淬灭单线态氧的能力相似,最小作用剂量均为0.005～0.05 g/L。     

连翘叶水提物保护线粒体及抗衰老研究

研究连翘叶水提物(FLA)对自由基所致的线粒体损伤的保护作用,并研究其抗氧化能力、活性氧的清除活性及抗衰老机制.采用硫代巴比妥酸(TBA)比色法测定丙二醛(MDA)含量、分光光度法测线粒体的肿胀度,并以还原型辅酶Ⅰ-氮蓝四唑-吩嗪硫酸甲酯(NADH-NBT-PMS)为超氧阴离子生成系统测定对超氧阴离子的清除能力.利用钼酸胺比色法、黄嘌呤氧化酶法、二硫代二硝基苯甲酸比色法、Fenton反应显色法分别测定FLA对小鼠肝匀浆过氧化氢酶(CAT)、超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)及抗羟自由基活力的影响.FLA可明显抑制线粒体的损伤及线粒体肿胀,并呈剂量依赖关系,清除超氧阴离子的能力显著,且能明显增强CAT、SOD、GSH-Px及抗羟自由基活力.FLA能通过清除活性氧自由基及提高抗氧化酶活力来保护线粒体,具有一定的抗氧化及抗衰老药用价值.     

蜂胶对氧自由基致鼠红细胞膜损伤的保护作用

应用脂质过氧化法和荧光探针法研究了蜂胶对氧自由基致鼠红细胞脱膜氧化损伤的保护作用。结果表明，蜂胶能显著地抑制氧自由基引发的鼠红细胞膜脂质过氧化反应，脂质过氧化产物丙二醛（MDA）含量显著下降，呈现量效关系。用Fenton反应产生的羟基自由基攻击红细胞膜后，膜脂的流动性显著下降，当红细胞膜经40μg/ml的蜂胶预先处理后，膜脂的流动性显著提高，表明蜂胶对氧自由基引发的鼠红细胞经损伤有显著的保护作用。     

Quenching Mechanism and Kinetics of Ascorbic Acid on the Photosensitizing Effects of Synthetic Food Colorant FD&C Red Nr 3

ABSTRACT:  The pH effect on the oxidative stability of ascorbic acid in the presence of food colorant FD&C Red Nr 3 during storage with or without light was investigated. The quenching mechanism and kinetics of ascorbic acid on the FD&C Red Nr 3 photosensitized oxidation in an aqueous system at 25 °C were also studied by measuring the degradation of ascorbic acid or depletion of headspace oxygen. Red Nr 3 had no influence on the oxidation of ascorbic acid under dark storage, but accelerated its oxidation rate under light storage. The oxidative stability of ascorbic acid decreased as the pH increased from 4 to 7 under light without FD&C Red Nr 3. The quenching rates of ascorbic acid on the singlet oxygen by measuring the degradation of ascorbic acid in the presence of Red Nr 3 under light storage were 1.53 ± 0.15 × 10⁸, 1.86 ± 0.25 × 10⁸, and 1.19 ± 0.12 × 10⁸ M⁻¹S⁻¹ at pH 4, 5.6, and 7, respectively.     

A Comparative Study on the Various In Vitro Assays of Active Oxygen Scavenging Activity in Foods

ABSTRACT: Various in vitro assays of active oxygen-scavenging activity in foods were compared by evaluating the activity of 13 food phenolic compounds and Trolox. 1,1-Diphenyl-2-picrylhydrazyl (DPPH)-HPLC and liposome oxidation methods were sensitive for these compounds, while deoxyribose oxidation and Randox kit methods were less sensitive. The deoxyguanosine oxidation method could not determine the activity since food polyphenols were pro-oxidative in this assay. Among these assays, the DPPH-HPLC method was shown to be the best since the liposome oxidation method is affected more by the structure of phenolic compounds than the DPPH-HPLC method.     

Effect of length and temperature of storage on the ESR signal from various bones in irradiated chicken carcasses

Eighty-four whole, fresh chicken carcasses were randomly allocated to three groups of 28. Half of the carcasses within each group acted as controls (0kGy) and the remainder received an average irradiation dose of 5.27 kGy. The carcasses were then stored at either +20°C, +5°C or -20°C for periods of 1, 3, 7, 14, 21, 28, 56, 84 or 128 days. Bones from four sites within the carcass (tibia, femur, rib and sternum) were removed and prepared for analysis by electron spin resonance (ESR) spectroscopy. The strength of the ESR signal induced in the bones as a result of gamma irradiation was determined by double integration of these signals. The site from which the bone was removed had a highly significant effect on the ESR signal intensity and this response was highly correlated with bone crystallinity. Neither storage time, temperature nor the interaction of these two variables significantly affected the ESR signal strength in the bones from the four sites within the carcass. Nevertheless, there was a tendency for the signal to decrease with time when the samples were stored in dried from at +20°C.     

高氧气调包装对鲜切莴苣呼吸和酶活性的影响

以空气包装的鲜切莴苣为对照,研究了60%O2﹢20%CO2﹢20%N2和100%O2高氧气调包装的鲜切莴苣在4℃贮藏14d期间呼吸强度和酶活性的变化。结果表明:高氧气调包装对鲜切莴苣呼吸和酶活性影响显著,60%O2﹢20%CO2﹢20%N2和100%O2高氧气调包装显著抑制鲜切莴苣的呼吸,且100%O2气调包装的抑制效果优于60%O2﹢20%CO2﹢20%N2气调包装;60%O2﹢20%CO2﹢20%N2和100%O2高氧气调包装促进SOD酶活性的上升、抑制O2-.生成量的增加和PPO、PAL酶活性的上升。     

活性氧对河岸葡萄叶圆片抗氧化酶活性的影响

研究了离体处理下活性氧对河岸葡萄叶圆片抗氧化酶活性的影响.结果表明,外源H2O2和Fe2++H2O2处理4 h可引起河岸葡萄叶圆片细胞膜透性明显增加,引起APOD,GPOD和SOD活性明显下降,但低浓度活性氧可不同程度引起叶圆片CAT活性增加;细胞膜透性与活性氧浓度呈显著正相关.