Reaction Products from Various Tocopherols with Trimethylamine Oxide and Their Antioxidative Activities

The structures of many reaction products obtained when various tocopherols (Toc’s) and trimethylamine oxide (TMAO) were treated in liquid paraffin under a nitrogen stream at 180°C, were determined and their antioxidative activities were investigated.

The reaction products (Toc dimers) isolated were as follows: α-tocopheryl ethane from α-Toc; 5-(γ-tocopheryloxy)-γ-Toc, 5-(γ-tocopheryl)-γ-Toc (two kinds) and α-tocopheryl ethane from γ-Toc; 5-(δ-tocopheryloxy)-δ-Toc from δ-Toc.

The two 5-(γ-tocopheryl)-γ-Toc’s are atropisomers of each other (TLC (Rf): 0.75, 0.45—benzene) and isomerization occurred within 20 min when they were treated under nitrogen at 180°C.

All Toc dimers, in particular 5-(γ-tocopheryloxy)-γ-Toc, have antioxidative activities and excellent synergism with TMAO in inhibiting the oxidation of lard kept in the dark at 60°C.     

Products Formed by Photosensitized Oxidation of Tocopherols

Two isomers of primary products formed by méthylene blue sensitized photooxidation of δ-tocopherol were separated by thin layer chromatography. The products were characterized by UV, IR and NMR spectra and were identified to be 8aS- and 8aR-hydroperoxy δ-tocopheryl dienones, respectively. On decomposition of the photooxidized α-, γ- and δ-tocopherols, to-copheryl quinone and tocopheryl quinone epoxide were formed. The effect of hydroperoxy 5-tocopheryl dienone on methyl linoleate peroxidation was examined. The hydroperoxy dienone formed by the photooxidation of tocopherols did not accelerate lipid peroxidation.     

Morphological and Chemical Differentiation between Tunisian Populations of Pinus halepensis,Pinus brutia,and Pinus pinaster

The lipid fraction of seeds from different pine species and populations was studied regarding total lipid content, fatty acid profile and vitamin E composition. The investigated seeds contained a high percentage of lipid (13.6 to 31.5 %). Lipid fractions were found to be rich in vitamin E, which varied significantly among species and populations. P. halepensis (Ph−Hn) showed the highest content of vitamin E (256.3 mg/kg of seeds) and the uppermost content of α-tocopherol (44 mg/kg). However, P. halepensis (Ph−Kas) was the richest in γ-tocopherol (204.9 mg/kg). Lipid fractions had a low content of δ-tocopherol (1.2 to 3.6 mg/kg. The highest content of γ-tocotrienol (∼18 %) was determined for P. halepensis (Ph−Dc and Ph−Hn). Thirteen fatty acids were identified by GC-FID with significant variation between the investigated species. The linoleic acid was the major fatty acid followed by oleic acid and palmitic acid. The chemical differentiation among species for the composition of fatty acids and vitamin E was confirmed by PCA. Significant correlations were observed between the content of vitamin E and fatty acids and ecological parameters of P. halepensis populations.     

Intracellular localization of tocopherol biosynthesis in calendula officinalis

[³H]Phytol was administered to protoplasts from Calendula officinalis leaves, and in the subcellular fractions the dynamics of labelling of 7-monomethyltocol, 8-monomethyltocol (δ-tocopherol), 7,8-dimethyltocol (γ-tocopherol) and 5,7,8-trimethyltocol (α-tocopherol) and related phytylquinones, as well as those of vitamin K₁, were determined. By condensation with homogentisic acid two isomeric methylphytylquinones (2-methyl-5-phytylbenzoquinone and 2-methyl-6-phytylbenzoquinone) were formed. These compounds were cyclized to 7- and 8-methyltocol, respectively, or methylated to yield 2,3-dimethyl-5-phytylbenzoquinone. The latter appeared to be cyclized to γ-tocopherol which could be methylated to α-tocopherol. The prenylation reaction took place in the chloroplasts and microsomes. Some monomethyltocols and methylphytylbenzoquinones as well as vitamin K₁ which appeared to be formed in microsomes may have been transported to chloroplasts and mitochondria.     

Lactones in the Flavor of Heated Pork Fat

Pork fat was heated at 160~170°C for 3 hr under bubbling with air, and the volatile compounds were collected in the cold trap. After the acidic compounds were removed from the volatile compounds by extraction with 3% aqueous sodium carbonate solution, lactones were obtained from the nonacidic compounds by saponification. Gas chromatographic analyses of lactones were carried out on the PEG-20M and Apiezon L packed columns, and then each lactone was fractionated by repeated gas chromatography. Each isolated lactone was identified by infrared spectrometry, and also three major lactones were identified by mass spectrometry. Consequently, γ-C₅—C₁₂ and δ-C₉, δ-C₁₀, δ-C₁₂ and δ-C₁₄ lactones were found in the flavor of heated pork fat. Gamma-lactones, especially γ-C₇, γ-C₈ and γ-C₉, were predominant in the flavor, and unsaturated lactones were not detected. Mechanisms for the formation of the lactones were discussed.     

Nitration of γ-tocopherol in plant tissues

Nitration of γ-tocopherol has been suggested to be an important mechanism for the regulation and detoxification of reactive nitrogen oxide species in animal tissues. To investigate whether this reaction does also occur in plants, reversed phase high-performance liquid chromatography (HPLC) and mass spectrometry (LC-MS) were used for analysis of 5-nitro-γ-tocopherol (5-NγT) in leaves and seeds. 5-nitro-γ-tocopherol (5-NγT) could be detected in an in vitro system where it was most likely generated by the reaction of γ-tocopherol with a nitric oxide radical. In vivo 5-NγT was identified in leaves of the Arabidopsis mutant line (vte4), which has insertion in the gene encoding γ-tocopherol methyltransferase and consequently lacks α-tocopherol and accumulates high levels of γ-tocopherol. Quantification of NO_x in leaves revealed that the vte4 mutant in comparison to wild type and the mutant vte1, which does not contain any tocopherol, has a reduced NO_x concentration. The level of 5-NγT in leaves of the vte4 mutant was shown to depend on the developmental stage and on the duration of light exposure. 5-NγT was also detectable in germinating seeds of Brassica napus, Nicotiana tabacum and Arabidopsis thaliana. These seeds have in common high γ-tocopherol contents. The rate of germination at two days after imbibition inversely correlated with the γ-tocopherol content of the seeds. The result suggests that γ-tocopherol or its respective derivative, 5-NγT, may prolong early development by reducing the level of NO_x.     

Antioxidant Effect of Tocopherols on Autoxidation of Milk Fat

Antioxidant activity of d-α-, dl-β-, d-γ- and d-δ-tocopherol was investigated with fatty acid methylester of milk fat from which unsaponifiable matter had been removed. Autoxidation was carried out at 50°C and its degree was indicated by peroxide value, α- or β-Tocopherol was more effective at lower concentrations (0.003 and 0.01%) than at higher concentrations (0.05, 0.1 and 0.5%). The antioxidant activity of γ- and δ-tocopherol was increased with the increase of tocopherol concentration within the range of 0.001 to 0.5%. The order of antioxidant activity of these tocopherols, which was compared in terms of the time to reach 30 meq of peroxide value, varied with the concentration; γ > β > δ > α at 0.001%, α > γ > β > δ at 0.003%, γ > δ > β > α at 0.01%, and δ > γ > β > α at the concentrations more than 0.05%. α-Tocopherol at the concentration of 0.003%, which corresponded to the concentration in original milk fat, was more effective than other tocopherols at the same concentration and α-tocopherol at other concentrations. Synergism due to the combination of β-, γ-, or δ-tocopherol with 0.003% of α-tocopherol was not observed.     

Studies on the Oxidation Mechanism of Vitamin E

α-Tocopherol model compound, 2,2,5,7,8-pentamethyl-6-hydroxychroman was oxidized under oxygen bubbling. Four oxidation products of 2-(γ,γ-dimethylallyl)-3,5,6-trimethyl-1,4-benzoquinone (B), 2,2,7,8-tetramethyl-5-formyl-6-hydroxychroman (C), trimer (D) and tocopherylethane (G) were identified, and spirodimer (E) was tentatively identified by TLC. Two of them, (B) and (C) have not been obtained in the oxidation of α-tocopherol model compound with p-quinone, alkaline ferricyanide and other compounds as oxidizing agent. A scheme of oxidation mechanism of α-tocopherol model compound was also proposed.     

Studies on the Changes of Meat Fats by Various Processings

Data are presented to show the gas chromatographic identification of a total of 18 saturated aliphatic γ- and δ-lactones obtained from melted beef depot fat, namely, δ-C₆, γ-C₇, γ-C₈, γ-C₉, and a homologous series of γ- and δ-lactones of the even-carbon numbers C₁₀ to C₁₆ and of smaller amount of the odd-carbon numbers C₁₁ to C₁₅. These lactones were isolated by steam distillation and silicic acid adsorption chromatography, and identified through gas chromatography and infrared spectroscopy.

Lactones obtained had a peach-like flavor, and it was suggested that lactones were important in heated beef fat as the flavor compounds.     

NADH-Dependent Glutamat Synthase in Euglena gracilis z

Novel antioxidative phenylpropanoid-substituted tocopherol derivatives, prunusols A and B, were isolated from the leaf wax of Prunus grayana Maxim., and their structures were fully characterized by spectroscopic and synthetic methods. Prunusols A and B were found to be the conjugates of γ-tocopherol and p-coumaric acid, which are diastereoisomers of each other. They showed almost the same antioxidative activity as α-tocopherol in a water/alcohol system measured by thiocyanate and TBA methods.     

Selective excretion of yolk-derived tocotrienols into the bile of the chick embryo

The aim of this study was to investigate the possibility of biodiscrimination between different forms of vitamin E during the development of the chick embryo. The vitamin E present in the initial yolk consisted of α-tocopherol (90%), (β+γ)-tocopherol (8%), α-tocotrienol (0.3%) and (β+γ)-tocotrienol (1.3%). In marked contrast, the vitamin E recovered from the bile of the day-16 embryo contained much higher proportions of α-tocotrienol (10%) and especially of (β+γ)-tocotrienol (42%). By the time of hatching, 56% of the vitamin E present in the bile was in the form of (β+γ)-tocotrienol. The residual yolk of the newly-hatched chick contained far greater proportions of α-tocotrienol (2.6%) and (β+γ)-tocotrienol (10%) than were present in the initial yolk. The results suggest that the liver of the embryo may selectively excrete tocotrienols as components of bile, whilst retaining the tocopherols within the hepatocytes. The increased proportions of tocotrienols in the residual yolk may result from the recycling of bile from the gall bladder to the yolk. The liver of the day-old chick contained α-tocopherol as the main form of vitamin E (90%) with only a small proportion (0.2%) of (β+γ)-tocotrienol. The α-tocopherol form was also the main vitamin E component in the brain (85%), heart (79%), lung (82%) and adipose tissue (91%) of the day-old chick. The present study suggests the occurrence of a high degree of biodiscrimination between tocopherols and tocotrienols during the development of the chick embryo.     

F2-Isoprostanes,tocopherols and normal pregnancy

This study investigates oxidative stress and antioxidants in normal human pregnancy and post-partum period. Thirty-seven healthy women with normal pregnancies were included. Both urinary and serum samples were collected throughout the pregnancy and post-partum period. Oxidative stress was estimated by measuring the reliable in vivo marker, namely 8-iso-prostaglandin F_2α (8-iso-PGF_2α, an F₂-isoprostane) and antioxidant status was evaluated by measuring α- and γ-tocopherol in serum samples. Pregnancy was associated with successively increased levels of 8-iso-PGF_2α with advancing gestational age. The median post-partum value corresponded to the values observed in early gestation and a significant decrease was observed from late pregnancy to the post-partum period. Lipid-adjusted α- and γ-tocopherol levels decreased with advancing gestational age. This longitudinal study suggests that mild oxidative stress is involved in normal human pregnancy.     

ZEIN DEGRADATION IN THE ENDOSPERM OF MAIZE SEEDS DURING GERMINATION

The pattern and sequence of zein degradation in the endosperm of germinating maize seeds were investigated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and by immunostaining Western blots with a monoclonal antibody to α-zein and polyclonal antibodies to β-, γ-, and δ-zeins. The results indicated: 1) the degradation of the predominant α-zein fraction (23.8 and 26.7 kD) started on the 5th day after germination (DAG) and continued gradually until the 10th DAG with a small amount remaining undegraded up to the 26th DAG; 2) β-zein (17 kD) began to be degraded on the 2d DAG, and the degradation of the 17 kD polypeptide was completed by the 7th DAG; 3) γ-zeins (27 and 18 kD) were the first zeins to be degraded, and the degradations of γ-zeinl (27 kD) and γ-zein2 (18 kD) were complete by the 3rd and 4th DAG, respectively; and 4) the degradation of δ-zein (10 kD) began on the 4th DAG and was complete by the 7th DAG. Based on these results, the following arrangement of zein polypeptides within the protein bodies is postulated, assuming that the proteolytic events start at the periphery and processed towards the core of the protein body: 1) γ-zeins are situated around the periphery of the protein bodies and are possibly a structural component of the protein body membrane or directly anchored in the membrane; 2) β-zein would be internal to γ-zeins; and 3) α-zein and δ-zein would be in the protein body core. This arrangement is largely consistent with published data on the immunocytochemical localization of zeins, and it indicates that the different classes of zein are not randomly organized within the matrix of the protein body.     

Syntheses of Unsaturated Lactones

6-Pentyl-α-pyrone, 6-propyl-α-pyrone and 4-decenoic acid-δ-lactone were prepared, and the nature of their flavors was investigated. Unsaturated lactones having the best flavorous nature as a butter or butter cake flavor among the lactones having double bond at various site, were 2-ene-δ-lactones which have a double bond at the α-position of the lactone ring and α-pyrones which have two double bonds at the α- and γ-positions. The flavor of 4-deceno-δ-lactone which has a double bond at the γ-position was the worst of them.     

Purification and Characterization of Lactobacillus bulgaricus Diaphorase

γ-Isomer of 1,2,3,4,5,6-hexachlorocyclohexane (BHC) showed greater decomposition on γ or UV irradiation of five isomers of BHC in crystalline state or in 2-propanol solution. The α- and δ-isomer of BHC and known la, 2a, 3e, 4e, 5e-pentachlorocyclohexane were separated from the irradiation product of crystalline γ-BHC. Four compounds were isolated from the irradiation product of γ-BHC in 2-propanol. Two compounds were tetrachloro-cyclohexenes (C₆H₆C1₄): γ-isomer (mp 86 ~87°C) and ?-isomer (mp 99 ~ 100°C). The other two were isomers of pentachlorocyclohexane (C₆H₇C1₅). One of them (mp 78 ~ 8.5°C) was consistent with known meso-1e,2a,3a,4a,5e isomer. The molecular structure of the other (mp 75°C) established by X-ray crystal structure analysis was 1α, 2α, 3α, 4β, 5α configuration or le 2a 3e 4e 5e conformation of CI atoms. A reaction mechanism was proposed that included a radical chain reaction and chlorine atom migration.     

Quenching Effect of Tocopherols on the Methyl Linoleate Photooxidation and Their Oxidation Products

The quenching effect of α-, γ- and δ-tocopherols on the methylene blue sensitized photo- oxidation of methyl linoleate was investigated, and the ¹O₂. quenching ability of tocopherols was determined. The ¹O₂ quenching rate constants for α-, γ- and δ-tocopherols in ethanol were estimated to be 2.6 × 10⁸ m^?1 sec^?1, 1.8 × 10⁸ m^?1 sec^?1 and 1.0 × 10⁸ m^?1 sec^?1, respectively. And the rate constants for the chemical reaction between each tocopherol and ¹O₂ were 6.6 × 10⁶ m^?1 sec^?1, 2.6 × 10⁶ m^?1 sec^?1 and 0.7 × 10⁶ m^?1 sec^?1 for α-, γ- and δ-tocopherols, respectively. The results show that α-tocopherol is the most effective compound toward ¹O₂ among the three tocopherols. The photooxidation of each tocopherol produced two peroxides which, after chemical reduction, were identified to be tocopherol hydroquinone by gas chromatography-mass spectrometry analysis. The photooxidation mechanism of these tocopherols was assumed to be different from that of autoxidation.     

A Facile Synthesis of Conjugated α-Methylene-δ-valerolactones

A facile synthetic method of variously substituted α-methylene-δ-valerolactones was devised and the growth inhibiting effects of synthesized samples were tested on rice seedlings. Alkylation of enamines (II) with methyl α-bromomethylacrylate (III) provided key intermediates, unsaturated keto esters (IV) which were readily converted to substituted α-methylene-δ-valerolactones (V) by a sequence involving saponification, reduction and lactonization. The growth inhibitory activities of the 6-membered α-methylene lactones synthesized closely paralleled that of α-methylene-γ-phenyl-γ-butyrolactone (XIII) which had already been shown in our previous paper to possess the inhibitory activity comparable to that of the natural inhibitors, heliangine and pyrethrosin.     

Isomerization of 1,3,4,5,6-Pentachlorocyclohexene-1 Isomers in Dimethyl Sulfoxide

In dimethyl sulfoxide, isomerization of 1,3,4,5,6-pentachlorocyclohexene-1 isomers was studied. Exchange of the allylic chlorine was indicated by the experiments with ³⁶Cl-labeled compounds. Isomerization at the position-3 proceeded faster than at the position-6. Initial reaction rates for the isomers decreased in the order: 34/56 > 35/46 (δ) > 346/5> 356/4 ? 36/45 (γ)^??. The specific rates at the initial stages increased by increasing the initial concentration of the substrates. After a prolonged reaction, the γ-isomer (36/45) was the most abundant component.

The isomers of the configuration: (34/56), (346/5), and (356/4) were isolated for the first time, on which PMR studies were performed.     

Studies on γ Globulin of Rice Embryo

All globulin components hitherto found in many species of seeds, α, β, γ and δ globulins, were identified in rice grain by ultracentrifugal experiments and gel-filtration chromatography. Among them, γ globulin was found to occur in high concentration in embryo and bran which were the most active parts in biological functions of rice grain. Then γ globulin was isolated from embryo by gel-filtration chromatography on a Sephadex G-200 column. Purified γ globulin was homogeneous in ultracentrifugal analysis and it was found to be insoluble in cold saline solution. On the other hand, α and β globulins were found to be more concentrated in endosperm with considerable heterogeneity.