Stoichiometric and kinetic studies on Ginkgo biloba extract and related antioxidants

Owing to increasing evidence showing the importance of lipid peroxidation in oxidative stress in vivo, the role and evaluation of antioxidants have received much attention. Ginkgo biloba extract (GBE), well-known as an efficient drug against diseases induced by free radicals, has been suggested to exert its effect by antioxidant action. A method was established to determine the activity of GBE as a hydrogen donor by stoichiometric and kinetic studies, and GBE was compared with several other antioxidants such as α-tocopherol, propyl gallate, and two kinds of flavonoids which are found in GBE, quercetin, and kaempferol. It was found that there were 6.62×10¹⁹ active hydrogens in 1g of GBE. Stoichiometric studies showed that one molecule of α-tocopherol reacted with one molecule of galvinoxyl radical. For quercetin, kaempferol and propyl gallate, the experimental stoichiometric numbers were 4.0, 1.9, and 3.1, respectively. The rates of reaction of antioxidants with galvinoxyl in ethanol were determined spectrophotometrically, using a stopped-flow technique. The second-order rate constant, k ₂, obtained at 25°C was 0.13 (g/l)⁻¹s⁻¹ for GBE and 5.9×10³, 2.1×10³, 1.2×10⁴, and 2.4×10³ M⁻¹s⁻¹ for quercetin, kaempferol, propyl gallate, and α-tocopherol, respectively. The second-order rate constant, k ₂′, on the molar basis of active hydroxyl groups in the tested substances obtained at 25°C decreased in the order of propyl gallate > α-tocopherol> quercetin>GBF∼kaempferol. This is the first study on GBE as an antioxidant which reports both stoichiometric and kinetic results.     

Mesoporous silica-based electrochemical sensor for simultaneous determination of honokiol and magnolol

A kind of mesoporous SiO₂ was synthesized using cationic surfactant as the structure-directing template. After that, the resulting mesoporous SiO₂ was used to modify the carbon paste electrode (CPE). The electrochemical behaviors of honokiol and magnolol were examined. In pH 6.5 phosphate buffer, two well-shaped oxidation peaks at 0.31 and 0.44 V were observed at the mesoporous SiO₂-modified CPE. Compared with the unmodified CPE, the mesoporous SiO₂-modified CPE remarkably enhances the oxidation peak currents of honokiol and magnolol. This suggests that mesoporous SiO₂ exhibits considerable surface enhancement effects to honokiol and magnolol. After optimizing the parameters such as pH value, amount of mesoporous SiO₂, and accumulation time, a sensitive and simple electrochemical method was proposed for the simultaneous determination of honokiol and magnolol. As to honokiol, the calibration curve is from 2.0 to 100.0 μg L⁻¹, and the limit of detection is 0.5 μg L⁻¹ (1.8 × 10⁻⁹ mol L⁻¹). For magnolol, the linear range is from 20.0 to 200.0 μg L⁻¹, and the limit of detection is 10.0 μg L⁻¹ (3.8 × 10⁻⁸ mol L⁻¹). Finally, the newly proposed method was successfully employed to determine honokiol and magnolol in Chinese traditional medicines.     

α-tocopherol oxidation mediated by superoxide anion (O 2 − ). II. Identification of the stable α-tocopherol oxidation products

The present paper describes the identification of two stable end products of α-tocopherol oxidation that were previously detected among the products of the reaction of α-tocopherol with superoxide anion (O ₂ ⁻ ) under aprotic conditions. One compound, previously designated compound A, was identified astrans-7-hydroxy-trans-8,8a-epoxy-α-tocopherone, and the other, designated compound B, was identified ascis-7-hydroxy-cis-8,8a-epoxy-α-tocopherone. It was also observed that under protic conditions (10% water in acetonitrile) the reaction of α-tocopherol with O ₂ ⁻ did not produce compounds A and B, but rather α-tocopheryl quinone, α-tocopherol dimer, α-tocopherol dihydroxy dimer, and the previously designated compound C. Compound C was identified in the present study as α-tocopheryl-quinone-2,3-epoxide.     

Ascorbate and phenolic antioxidant interactions in prevention of liposomal oxidation

Efficient prevention of membrane lipid peroxidation by vitamin E (α-tocopherol) may involve its regeneration by vitamin C (ascorbate). Conceivably, the efficacy of antioxidants designed as therapeutic agents could be enhanced if a similar regeneration were favorable; thus, a model membrane system was developed which allowed assessment of interaction of phenolic antioxidants with ascorbate and ascorbyl-6-palmitate. Ascorbate alone (50–200 μM) potentiated oxidation of soybean phosphatidylcholine liposomes by Fe²⁺/histidine-Fe³⁺, an effect which was temporally related to reduction of Fe³⁺ generated during oxidation. Addition of 200 μM ascorbate to α-tocopherol-containing liposomes (0.1 mol%) resulted in marked, synergistic protection. Accordingly, in the presence but not absence of ascorbate, α-tocopherol levels were maintained relatively constant during Fe²⁺/histidine-Fe³⁺ exposure. Probucol (4,4′-[(1-methylethylidine)bis(thio)]bis[2,6-bis(1,1-dimethylethyl)]phenol), and antioxidant which prevents oxidation of low density lipoproteins, and its analogues MDL 27,968 (4,4′-[(1-methylethylidene)bis(thio)]-bis[2,6-dimethyl]phenol) and MDL 28,881 (2,6-bis(1,1-dimethylethyl)-4-[(3,7,11-trimethyldodecyl)thio]phenol) prevented oxidation but exhibited no synergy with ascorbate. Ascorbyl-6-palmitate itself was an effective antioxidant but did not interact synergistically with any of the phenolic antioxidants. Differential scanning calorimetry revealed significant differences among the antioxidants in their effect on the liquid-crystalline phase transition of dipalmitoyl phosphatidylcholine (DPPC) liposomes. Both α-tocopherol and MDL 27,968 significantly reduced the phase transition temperature and the enthalpy of the transition. MDL 28,881 had no effect while probucol was intermediate. The potential for ascorbate or its analogues to interact with phenolic antioxidants to provide a more effective antioxidant system appears to be dictated by structural features and by the location of the antioxidants in the membrane.     

Extra Virgin Olive Oil Components and Oxidative Stability from Olives Grown in Tunisia

The effects of the contents of lipids, pigments, α-tocopherol and phenols were studied in relation to the antioxidant capacity of five virgin olive oils obtained from five olive cultivars planted in Tunisia (Arbequina, Koroneiki, Leccino, Oueslati and Chemchali). The antioxidant capacities were evaluated by two different radical scavenging activities: radical scavenging activity by the DPPH assay (RSA-DPPH) and total antioxidant status by the ABTS test (TAA-ABTS). The highest contents of antioxidant compounds (75.96, 10.34, 6.32, 15.39 and 241.52 mg kg⁻¹ for oleic acid, O/L ratio, carotenes, chlorophylls and total phenols, respectively) were found for the Koroneiki cultivar except for α-tocopherol and o-diphenols, which had the highest contents (369 and 160.7 mg kg⁻¹, respectively) in the Leccino and Chemchali cultivars (cvs). Furthermore, the highest antioxidant capacity in virgin olive oil was observed in the Koroneiki cultivar (0.24 mmol TE kg⁻¹) followed by the Chemchali and Leccino cvs (0.22 and 0.13 mmol TE kg⁻¹) for the TAA-ABTS test. However, the RSA-DPPH activity was higher for the Chemchali cultivar (19.9%) than for the Koroneiki and Leccino cvs (18.4 and 13.5%, respectively). Correlation between these capacities and the oil composition revealed that they were mainly influenced by the carotene content, followed by chlorophyll and phenolic contents where the ABTS test was more pronounced. Then, the antioxidant capacity of the virgin olive oils was correlated with polar components and the lipid profile which are important for its shelf life.     

Effect of natural antioxidants in virgin olive oil on oxidative stability of refined,bleached, and deodorized olive oil

The factors influencing the oxidative stability of different commercial olive oils were evaluated. Comparisons were made of (i) the oxidative stability of commercial olive oils with that of a refined, bleached, and deodorized (RBD) olive oil, and (ii) the antioxidant activity of a mixture of phenolic compounds extracted from virgin olive oil with that of pure compounds andα-tocopherol added to RBD olive oil. The progress of oxidation at 60°C was followed by measuring both the formation (peroxide value, PV) and the decomposition (hexanal and volatiles) of hydroperoxides. The trends in antioxidant activity were different according to whether PV or hexanal were measured. Although the virgin olive oils contained higher levels of phenolic compounds than did the refined and RBD oils, their oxidative stability was significantly decreased by their high initial PV. Phenolic compounds extracted from virgin olive oils increased the oxidative stability of RBD olive oil. On the basis of PV, the phenol extract had the best antioxidant activity at 50 ppm, as gallic acid equivalents, but on the basis of hexanal formation, better antioxidant activity was observed at 100 and 200 ppm.α-Tocopherol behaved as a prooxidant at high concentrations (>250 ppm) on the basis of PV, but was more effective than the other antioxidants in inhibiting hexanal formation in RBD olive oil.o-Diphenols (caffeic acid) and, to a lesser extent, substitutedo-diphenols (ferulic and vanillic acids), showed better antioxidant activity than monophenols (p- ando-coumaric), based on both PV and hexanal formation. This study emphasizes the need to measure at least two oxidation parameters to better evaluate antioxidants and the oxidative stability of olive oils. The antioxidant effectiveness of phenolic compounds in virgin olive oils can be significantly diminished in oils if their initial PV are too high.     

Chemical composition and antioxidant activity of extracts from <Emphasis Type="Italic">Daphne gnidium</Emphasis> L.

Members of the Daphne genus have been of interest owing to their excellent medicinal value. In this work, we describe the results of phytochemical analysis and the antioxidant activity of the methanol extracts from leaves and stems of D. gnidium L. grown wild in Sardinia, Italy. Four coumarins (daphnetin, daphnin, acetylumbelliferon, and daphnoretin), nine flavonoids (apigenin, luteolin, quercetin, orientin, isoorientin, luteolin 7-O-glucoside, apigenin 7-O-glucoside, genkwanin, and 5-O-β-d-primeverosyl genkwanine), and α-tocopherol were isolated. We investigated the ability of the two extracts and five pure compounds (daphnetin, daphnoretin, apigenin, luteolin, and α-tocopherol) to protect linoleic acid against free radical attack in simple in vitro systems by autoxidation and iron- or EDTA-mediated oxidation. Pure compounds were the most active antioxidants. During autoxidation, daphnetin, luteolin, and α-tocopherol were effective at a molar ratio of 1∶1600, 1∶2500, and 1∶2000, respectively. Daphnoretin was active only at high concentrations. During the iron-catalyzed oxidation of linoleic acid, all the materials tested showed activity in the following order: luteolin>daphnetin>α-tocopherol >leaf extract>stem extract>daphnoretin. Apigenin was not active in any of the experimental systems used.     

Arachidonic acid autoxidation in an aqueous media effect of α-tocopherol,cysteine and nucleic acids

The autoxidation of arachidonic acid dispersed in aqueous media was evaluated simultaneously with and without different agents, e.g., α-tocopherol at different concentrations, cysteine, DNA and RNA. The autoxidation rate of arachidonic acid was evaluated by quantitative gas liquid chromatography (GLC) determination of the unoxidized acid and by spectrophotometric measurement of conjugated dienes. α-Tocopherol exhibited a prooxidant activity at concentrations of 1.25 × 10⁻⁴ M and 1.25 × 10⁻⁵ M and a weak antioxidant activity at a concentration of 1.25 × 10⁻⁶ M. Cysteine showed antioxidant activity and greatly reduced the prooxidant activity of α-tocopherol. DNA and RNA had no effect in either case. α-Tocopherol oxidation was followed by high pressure liquid chromatography (HPLC). The prooxidant effect was accompanied by a rapid oxidation of α-tocopherol, except in the presence of cysteine, which prevented the oxidation of α-tocopherol.     

α-tocopherol oxidation mediated by superoxide anion (O 2 − ) I. Reactions in aprotic and protic conditions

The reaction of α-tocopherol (α-T) with superoxide anion (O ₂ ⁻ ) in both dry acetonitrile and in aqueous acetonitrile solution is described. The O ₂ ⁻ was generated by the electrochemical reduction of molecular oxygen in acetonitrile, using tetrabutylammonium bromide as an electrolyte. α-T was reacted with O ₂ ⁻ either in dry acetonitrile or in a 10% aqueous acetonitrile solution. In dry acetonitrile, α-T was oxidized to a very unstable primary intermediate, which was further oxidized to a secondary, more stable intermediate. The formation of the secondary intermediate depended upon the presence of molecular oxygen. This intermediate readily converted into two compounds in equimolar amounts (designated A and B). The primary, very unstable intermediate was readily reduced again to α-T by treatment with LiAlH₄ or ascorbic acid. However, the secondary intermediate or the stable oxidation products could not be reduced to α-T. In the 10% aqueous acetonitrile, α-T was oxidized to α-tocopheryl quinone, α-tocopherol dimer and α-tocopherol dihydroxy dimer, and an unknown compound. In the aqueous medium, no intermediates were formed by the action of O ₂ ⁻ . The results of this study indicate that the reaction of α-T with O ₂ ⁻ under aprotic conditions is different from that observed under protic conditions.     

Peroxide dependent and independent lipid peroxidation: Site-specific mechanisms of initiation by chelated iron and inhibition by α-tocopherol

Peroxidation of linoleic acid (LA) was catalyzed by Fenton reagent (H₂O₂, and Fe²⁺) in positively charged tetradecyltrimethylammonium bromide (TTAB) micelles, but not in negatively charged sodium dodecylsulfate (SDS) micelles. However, more hydroxyl radicals formedvia the Fenton reaction were trapped byN-t-butyl-α-phenylnitrone (PBN) in SDS micelles than in TTAB micelles. Generation of linoleic acid alkoxy (LO) radicals by Fe²⁺ via reductive cleavage of linoleic acid hydroperoxide (LOOH) resulted in peroxidation of LA and formation of PBN-LO· adducts in SDS micelles, but not in TTAB micelles. This LOOH dependent lipid peroxidation could be catalyzed in TTAB micelles in the presence of a negatively charged iron chelator, nitrilotriacetic acid (NTA). LO radicals formed by the LOOH dependent Fenton reaction were also trapped by PBN at the surface of TTAB micelles in the presence of NTA, but not in its absence. The consumption of a spin probe, 16-(N-oxyl-4,4′-dimethyloxazolidin-2-yl)stearic acid (16-NS) during the LOOH dependent Fenton reaction in the presence of NTA was higher in TTAB micelles of LA than in those of lauric acid (LauA), although the rates and amounts of LO radicals formed in the two types of fatty acid micelles were similar. The rates of 5-NS consumption in LA and LauA micelles were almost the same, and were lower than the rate of 16-NS in LA micelles. NTA-Fe²⁺ initiated peroxidation of LA in TTAB micelles without a lag time in the presence of LOOH, but after a lag period, peroxidation occurred without LOOH. α-Tocopherol inhibited peroxidation of LA catalyzed by Fenton reagent by scavenging OH radicals in TTAB micelles. In contrast, α-tocopherol enhanced free Fe²⁺ induced LOOH dependent lipid peroxidation through the regeneration of Fe²⁺ in SDS micelles. However, it inhibited NTA-Fe²⁺ induced LOOH dependent lipid peroxidation in TTAB micelles. The rate and amount of α-tocopherol oxidized by the Fe²⁺ induced, H₂O₂ dependent Fenton reaction were almost the same in TTAB micelles of LA and LauA. The oxidation of α-tocopherol by the NTA-Fe²⁺ induced, LOOH dependent Fenton reaction was greater and faster in LA micelles than in LauA micelles, although the rates of LO radical production in the two types of micelles were the same. During NTA-Fe²⁺ induced, LOOH dependent lipid peroxidation, α-tocopherol inhibited more effectively the consumption of 16-NS than 5-NS. The results are discussed in relation to the location of iron, the unsaturated bonding region of LA, the OOH group of LOOH, the radical trapping site of PBN, the spin sites of 5-NS and 16-NS, and the phenolic hydroxyl group of α-tocopherol in micelles with different charges. Based on a paper presented at the Symposium on Metals and Lipid Oxidation, held at the AOCS Annual Meeting in Baltimore, MD, April 1990.     

Peroxyl and hydroxyl radical scavenging activity of some natural phenolic antioxidants

The autoxidation of linoleic acid dispersed in an aqueous media and the antioxidant effect of hydroxytyrosol, oleuropein, caffeic acid and tyrosol were studied. Linoleic acid autoxidation rate was estimated by the increase of conjugated diene level and by the decrease of linoleic acid content in the samples. The phenolic compounds exhibited an antioxidant activity which increased in the order: tyrosol < caffeic acid < oleuropein < hydroxytyrosol. The analysis of the hydroperoxide isomers pointed out that hydroxytyrosol, oleuropein and caffeic acid at a concentration of 10⁻⁴M inhibited the formation oftrans- trans isomers in the increasing order: caffeic acid < oleuropein < hydroxytyrosol. This inhibition could be related to the ability of phenolic compounds to scavenge peroxyl radical. Tyrosol did not inhibit the formation oftranstrans isomers. Phenolic compounds were degraded as a consequence of their antioxidant activity and their degradation rate was positively correlated to their antioxidant efficacy. These phenolic compounds, at a concentration of 6 × 10⁻³M, also scavenged hydroxyl radical, with an efficiency which increased in the order: tyrosol < hydroxytyrosol < oleuropein < caffeic acid. Polar substituents at the para position, such as in caffeic acid and oleuropein, were correlated with higher hydroxyl radical quenching ability.     

Antioxidant activities of tocopherols on Fe2+-ascorbate-induced lipid peroxidation in lecithin liposomes

The antioxidant activities of 4 tocopherols, tocol, and a water-soluble model analog of α-tocopherol were compared. Egg lecithin liposomes were used and oxidation was catalyzed by Fe²⁺-ascorbate. The activities decreased in the order α->β->γ->δ-tocopherol>tocol, in agreement with their potencies in vivo. The water-soluble analog was the least effective. Activity depended on the molar ratio of antioxidant to unsaturated lipid, with one molecule each of the α-, β-, γ-, δ-tocopherol and tocol capable of protecting, respectively, 220, 120, 100, 30 and 20 molecules of polyunsaturated fatty acid. The mechanism of possible antioxidant effect of the compounds used is discussed.     

Characterization of Aegean Olive Oils by Their Minor Compounds

This study presents a combined approach of establishing cultivar differences between Aegean olive oils, obtained from economically important olive oil producing cultivars (cv. Ayvalik and Memecik), based on chemometric evaluation of their content and in particular composition of the minor compounds. Evaluation of minor compounds with principal component analysis and linear discriminant analysis (LDA) indicated differentiation according to the cultivars. LDA produced a 100% correct group classification. Moreover, stigmasterol, apparent β-sitosterol and total sterols were found to have the highest discriminating power. Memecik oils were characterized by the highest content of antioxidant compounds (α-tocopherol, phenolic compounds and total phenolic compounds). On the other hand, Ayvalik oil had the highest level of total sterols. The data were analyzed statistically to evaluate the differences according to variety and crop season. The minor compounds of Ayvalik and Memecik oils presented statistically significant differences (p < 0.01) according to variety, except for the hydroxytyrosol and clerosterol content. The amount of α-tocopherol, total phenolic compounds, apparent β-sitosterol and total sterols varied with respect to crop season. A good correlation was observed between the amount of α-tocopherol, total phenolic compounds, apparent β-sitosterol and total sterols and some climatic variables.     

Tocotrienols from palm oil: Electron spin resonance spectra of tocotrienoxyl radicals

The major vitamin E components present in palm oil, viz. α-tocopherol, α⁻, ψ-and δ-tocotrienols, have been isolated and their structures verified by the NMR spectra of their acetate and succinate derivatives. Oxidation of γ-and δ-tocotrienols with alkaline K₃Fe(CN)₆ gave isolable dimeric species, which were studied by¹³C NMR. Free radicals generated from the monomeric and dimeric tocotrienols were investigated using ESR spectroscopy. The distinction between antioxidant activity and antioxidant capacity of vitamin E isomers is discussed.     

Antioxidant activity of flavonol aglycones and their glycosides in methyl linoleate

The antioxidant activities of the flavonol aglycones, quercetin and myricetin, and their selected glycosides were compared in bulk methyl linoleate oxidized at 40°C. Methyl linoleate hydroperoxide formation, hydroperoxide isomer distribution, and ketodiene formation were followed by using high-performance liquid chromatography (HPLC) analysis. The aglycones, quercetin and myricetin, were consistently more active in bulk methyl linoleate than their glycosides and more active than α-tocopherol at 500 and 1000 μM. At 50 μM, the order of activity was myricetin > α-tocopherol > quercetin, and the order of activity of quercetin and its derivatives was quercetin > quercitrin > isoquercitrin > rutin. Myricitrin was slightly less active than myricetin. The sugar moiety was shown to have a marked effect on the antioxidant activity of flavonols. The rhamnoside derivatives, quercitrin and myricitrin, both possessed activity close to that of their corresponding aglycones. The different activities of glycosides could be partly explained by different solubilities and by differences in oxidizability of glycosides containing a monosaccharide or disaccharide at the C₃ position. The effect on hydroperoxide isomer distribution indicates that α-tocopherol was a more effective hydrogen donor than flavonoids, although flavonoids were more effective in inhibiting oxidation of methyl linoleate.     

Antioxidant Properties of Phenolic Lignin Model Compounds

Antioxidant activities of phenolic lignin model compounds were determined by measurements of inhibition rate constants (k_inh) during inhibited peroxidation of styrene in chlorobenzene initiated by azobisisobutyrylnitrile with known rates of initiation (R_i). The number of peroxyl radicals trapped by each antioxidant, the stoichiometric factors (n), were determined by comparison with pentamethyl-hydroxychroman, PMHC, n = 2. Monomeric lignin models, 4-propylguaiacol (1), eugenol (2), isoeugenol (3), coniferyl alcohol (4), coniferyl aldehyde (5), and 4-allyl-2,6-dimethoxyphenol (6) were all more active antioxidants than the commercial inhibitor 2,6-di-tert-butyl-4-methylphenol (BHT). Two dimer models, bis(2-hydroxy-3-methoxyl-5-allylphenyl)methane (7) and 2,2′-dihydroxy-3,3′-dimethoxy-5,5′-dimethoxymethylbiphenyl (8) and a synthetic tetramer, bis[2-hydroxy-5-(2′-hydroxy-3′-methoxy-5′-methylbenzyl)-3-methoxyphenyl]methane (9) were more active antioxidants. The overall relative activity was tetramer > dimers > monomers > BHT. The stoichiometric factors of 1 to 6 ranged from 1.6 to 1.7 compared to PMHC. The n factors for 7, 8, and 9, showed an additive effect per phenolic hydroxyl. Phenolic groups in lignin may protect lignin-containing pulps and paper against damaging free radical peroxidation.     

Vitamin E deficiency in dogs does not alter preferential incorporation ofRRR-α-tocopherol compared withall rac-α-tocopherol into plasma

The plasma and lipoprotein transport ofRRR andall rac-α-tocopherols, labeled with different amounts of deuterium [2R,4′R,8′R-α-[5-C²H₃]tocopheryl acetate (d ₃ RRR-α-tocopheryl acetate] and 2RS, 4′RS, 8′RS-α-[5,7-(C²H₃)₂]tocopheryl acetate (d ₅ all rac-α-tocopheryl acetate), was studied in adult beagle dogs that had been fed a vitamin E-deficient (−E; two dogs) or supplemented (+E; two dogs) diet for two years. We set out to test the hypothesis that the activity of the hepatic tocopherol binding protein (which is thought to preferentially incorporateRRR-α-tocopherol into the plasma) is up-regulated by vitamin E deficiency. Labeled α-tocopherols increased and decreased similarly in plasma of both −E and +E dogs. Irrespective of diet,d ₃ RRR-α-tocopherol was preferentially secreted in plasma. Thus, vitamin E deficiency in dogs does not markedly increase the apparent function of the hepatic tocopherol binding protein. We also studied vitamin E transport in a German Shepherd dog with degenerative myelopathy (DM). Based on the coincident appearance ofd ₃ RRR-α-tocopherol in plasma and chylomicrons, we suggest that the abnormality in DM may be associated with abnormal vitamin E transport resulting from an impaired function of the hepatic tocopherol binding protein.     

Rate constants for quenching singlet oxygen and activities for inhibiting lipid peroxidation of carotenoids and α-tocopherol in liposomes

The ¹O₂ quenching rate constants (k _Q ) of α-tocopherol (α-Toc) and carotenoids such as β-carotene, astaxanthin, canthaxanthin, and lycopene in liposomes were determined in light of the localization of their active sites in membranes and the micropolarity of the membrane regions, and compared with those in ethanol solution. The activities of α-Toc and carotenoids in inhibiting ¹O₂-dependent lipid peroxidation (reciprocal of the concentration required for 50% inhibition of lipid peroxidation: [IC₅₀]⁻¹) were also measured in liposomes and ethanol solution and compared with their k _Q values. The k _Q and [IC₅₀]⁻¹ values were also compared in two photosensitizing systems containing Rose bengal (RB) and pyrenedodecanoic acid (PDA), respectively, which generate ¹O₂ at different sites in membranes. The k _Q values of α-Toc were 2.9×10⁸M⁻¹s⁻¹ in ethanol solution and 1.4×10⁷ M⁻¹s⁻¹ (RB system) or 2.5×10⁶ M⁻¹s⁻¹ (PDA system) in liposomes. The relative [IC₅₀]⁻¹ value of α-Toc in liposomes was also five times higher in the RB system than in the PDA-system. In consideration of the local concentration of the OH-group of α-Toc in membranes, the k _Q value of α-Toc in liposomes was recalculated as 3.3×10⁶ M⁻¹s⁻¹ in both the RB and PDA systems. The k _Q values of all the carotenoids tested in two photosensitizing systems were almost the same. The k _Q value of α-Toc in liposomes was 88 times less than in ethanol solution, but those of carotenoids in liposomes were 600–1200 times less than those in ethanol solution. The [IC₅₀]⁻¹ value of α-Toc in liposomes was 19 times less than that in ethanol solution, whereas those of carotenoids in liposomes were 60–170 times less those in ethanol solution. There were no great differences (less than twice) in the k _q and [IC₅₀]⁻¹ values of any carotenoids. The k _Q values of all carotenoids were 40–80 times higher than that of α-Toc in ethanol solution but only six times higher that of α-Toc in liposomes. The [IC₅₀]⁻¹ values of carotenoid were also higher than that of α-Toc in ethanol solution than in liposomes, and these correlated well with the k _Q values.     

Extraction and identification of antioxidants from the spice Aframomum danielli

Successive extractions with diethyl ether and methanol of the whole seeds of the spice Aframomum danielli yielded diethyl ether extract (ADEE), 13.9%, and methanol extract (ADM), 3.4%, respectively. Similarly, reextraction of the defatted seeds of A. danielli successively with diethyl ether and methanol yielded extracts DFADEE (7.9%) and DFADM (6.7%), respectively. When these extracts were added to refined peanut oil (PNO) at 200 ppm, they showed good antioxidative effects. The percentage antioxidant effectiveness (AE) values were as follows: DFADM (87.3) > ADM (85.3) > ADEE (83.4)=tert-butyl hydroquinone (83.4) on day 20 of storage in an oven maintained at 65±1°C. Generally, antioxidant extracts prepared from A. danielli were also more effective than butylated hydroxytoluene and α-tocopherol in stabilizing refined PNO. Antioxidant components of A. danielli were tentatively identified as phenolic compounds of the trihydroxy type with reducing properties. All extracts prepared from A. danielli showed strong ultraviolet-absorbing characteristics, and methanol was a good extracting solvent.     

Isolation of a trimer of α-tocopherol from mammalian liver

Evidence is presented for the formation in mammalian liver of a trimeric metabolite of α-tocopherol. This compound has been shown to be identical to a trimer produced by oxidation of α-tocopherol with alkaline K₃Fe(CN)₆. In addition, confirmation was obtained for the occurrence in vivo of a dimeric metabolite reported previously. These compounds, together with tocopheryl-p-quinone, are postulated to arise from reactions with lipid-free radicals or peroxides in the course of the antioxidant action of vitamin E.