Determination of tocopherols as lipid antioxidants in vegetable oils and animal fats

The content of vitamin E (tocopherols) in natural vegetable oils and fats has been determined by microcalorimetry. This technique belongs to kinetic methods for determining vitamin E, which are based on the capacity of tocopherol to inhibit liquid-phase radical oxidation reactions. It has been shown on a model reaction of initiated oxidation of cumene that fatty oils inhibit the radical reaction with an induction period proportional to the content of tocopherols in oil. From experimental curves, the content of tocopherols in oils obtained by different technological methods has been estimated. It has been shown that the amount of tocopherols is an indicator of the oil quality; therefore, the method proposed can be used to control the way of oil manufacture, oil quality, and the presence of synthetic antioxidants.     

A review of characterization of tocotrienols from plant oils and foods

Tocotrienols, members of the vitamin E family, are natural compounds found in a number of vegetable oils, wheat germ, barley and certain types of nuts and grains. Vegetable oils provide the best sources of these vitamin E forms, particularly palm oil and rice bran oil contain higher amounts of tocotrienols. Other sources of tocotrienols include grape fruit seed oil, oats, hazelnuts, maize, olive oil, buckthorn berry, rye, flax seed oil, poppy seed oil and sunflower oil. Tocotrienols are of four types, viz. alpha (α), beta (β), gamma (γ) and delta (δ). Unlike tocopherols, tocotrienols are unsaturated and possess an isoprenoid side chain. A number of researchers have developed methods for the extraction, analysis, identification and quantification of different types of vitamin E compounds. This article constitutes an in-depth review of the chemistry and extraction of the unsaturated vitamin E derivatives, tocotrienols, from various sources using different methods. This review article lists the different techniques that are used in the characterization and purification of tocotrienols such as soxhlet and solid–liquid extractions, saponification method, chromatography (thin layer, column chromatography, gas chromatography, supercritical fluid, high performance), capillary electrochromatography and mass spectrometry. Some of the methods described were able to identify one form or type while others could analyse all the analogues of tocotrienol molecules. Hence, this article will be helpful in understanding the various methods used in the characterization of this lesser known vitamin E variant.     

Durum wheat by-products as natural sources of valuable nutrients

This review reports the use of wheat milling by-products for the extraction of high quality oil and vitamin E including our results on the exploitation of durum wheat bran as a valuable source of important healthful compounds. Wheat oil can be used as an ingredient in food, pharmaceutical or cosmetic preparations because it contains important bioactive compounds such as vitamin E, carotenoids and unsaturated fatty acids. Different methods are used for oil recovery from plant materials, such as solvent extraction, mechanical pressing or the eco-friendly supercritical carbon dioxide (SC-CO₂) extraction technology. By using SC-CO₂, we obtained an oil from durum wheat (Triticum durum Desf.) bran and optimized the extraction conditions to increase oil and vitamin E yields. Wheat bran, which is composed of pericarp, aleurone layer and germ, is discarded during the early stages of durum wheat milling processes to obtain a final product (semolina) that is stable over time. Maximum oil and vitamin E yields were obtained when a durum wheat bran matrix with particle size of ~30 mesh and a moisture content of 2.6 % was used. The optimal conditions for oil extraction were: 300–350 bar, 60–70 °C, and 4 l min^?1 gaseous CO₂ flow rate for 1 h. The chemical composition (vitamin E forms, carotenoids, quinones, lipids and fatty acids) of the SC-CO₂ extracted oil was analyzed and compared to that of the oil extracted by Soxhlet using hexane as solvent. The findings here reported highlight the importance of durum wheat bran as a rich source of valuable natural nutrients.     

Tocopherol and tocotrienol accumulation during development of caryopses from barley (Hordeum vulgare L.)

Tocotrienols are lipophilic antioxidants belonging to the tocochromanols, better known as vitamin E. Although present in cereal grains in high quantities not much is known about their function in plants. In a detailed study the temporal and spatial accumulation of tocotrienols and tocopherols during grain development in two barley cultivars was analyzed. Tocochromanols and lipids accumulated in parallel until 80% of the final dry weight of the kernels was reached. Later on the tocochromanol content did not change while the lipid content decreased. Generally, only about 13% of the tocochromanols were found in the germ fraction, whereas the pericarp fraction contained about 50% and the endosperm fraction about 37% of the tocochromanols. Altogether, about 85% of the tocochromanols were tocotrienols in both cultivars. In case of the tocopherols about 80% were found in the germ fraction and the remaining 20% in the pericarp fraction. Tocotrienols were almost equally present in the pericarp and the endosperm fraction. Individual forms of tocopherols and tocotrienols accumulated with different kinetics during barley grain development. The differences in distribution and accumulation indicate different functions of the individual tocochromanols during grain development.     

Lipase catalyzed deacidification of tocopherol-rich distillates obtained from natural Vitamin E sources

Taking into account the great interest of consumers for high purity natural vitamin E preparations, it is highly desirable to develop an efficient process for recovering Vitamin E from vegetable oil deodorizer distillates (VODDs) with reduction in costs and better use of energy. The process described herein involves a series of treatment steps, namely desaromatization of VODD, concentration of tocopherols by molecular distillation at high vacuum, lipase catalyzed ester synthesis, and brightening of the resulting tocopherol-rich concentrate with an ethanol (96%):water 60:40 mixture which are similar to conventional processes but the purpose and order of execution of some of them are clearly different. Moreover, some treatment steps were shortened or directly removed. By this process a fully translucent and fluid product was obtained which had tocopherol richness near to the standardized level of 50% concentration, acidity around 1% and a content of emulsifying mono- and diglycerides of 20%. The mixture of mono- and diglycerides that result from the enzymatic process could facilitate absorption of tocopherols by the body and thereby enhancing the biological activity of these compounds. Because all these facts the process presented is feasible as a new industrial purification method of tocopherols from VODDs.     

Metabolic redesign of vitamin E biosynthesis in plants for tocotrienol production and increased antioxidant content

Tocotrienols are the primary form of vitamin E in seeds of most monocot plants, including cereals such as rice and wheat. As potent antioxidants, tocotrienols contribute to the nutritive value of cereal grains in human and livestock diets. cDNAs encoding homogentisic acid geranylgeranyl transferase (HGGT), which catalyzes the committed step of tocotrienol biosynthesis, were isolated from barley, wheat and rice seeds. Transgenic expression of the barley HGGT in Arabidopsis thaliana leaves resulted in accumulation of tocotrienols, which were absent from leaves of nontransformed plants, and a 10- to 15-fold increase in total vitamin E antioxidants (tocotrienols plus tocopherols). Overexpression of the barley HGGT in corn seeds resulted in an increase in tocotrienol and tocopherol content of as much as six-fold. These results provide insight into the genetic basis for tocotrienol biosynthesis in plants and demonstrate the ability to enhance the antioxidant content of crops by introduction of an enzyme that redirects metabolic flux.     

Beyond vitamin E supplementation: an alternative strategy to improve vitamin E status

Vitamin E has many reported health effects and is recognized as the most important lipid-soluble, chain-breaking antioxidant in the body. Vitamin E has also been reported to play a regulatory role in cell signalling and gene expression. Epidemiological studies show that high blood concentrations of vitamin E are associated with a decreased risk of cardiovascular diseases and certain cancers. Yet, high doses of supplemental vitamin E have been associated with an elevated risk of heart failure and all-cause mortality. Therefore, establishing alternative strategies to improve vitamin E status without potentially increasing mortality risk may prove important for optimal nutrition. To identify dietary phenolic compounds capable of increasing blood and tissue concentrations of vitamin E, selected polyphenols were incorporated into standardized, semi-synthetic diets and fed to male Sprague-Dawley rats for 4 weeks. Blood plasma and liver tissue concentrations of alpha-T and gamma-Twere determined. The flavanols (+)-catechin and (-)-epicatechin, the flavonol quercetin, and the synthetic preservative butylated hydroxytoluene (BHT) markedly elevated the amount of alpha-T in plasma and liver. The sesame lignan sesamin and cereal alkylresorcinols substantially increased the concentrations of gamma-T, but not alpha-T, in the liver. Sesamin also increased gamma-T concentrations in plasma. In order to study the impact of selected polyphenols on the enzymatic degradation of vitamin E, HepG2 cells were incubated together with phenolic compounds in the presence of tocopherols and the formation of metabolites was determined. Sesamin, at concentrations as low as 2 microM, almost completely inhibited tocopherol side-chain degradation and cereal alkylresorcinols inhibited it, dose-dependently (5-20 microM), by 20-80%. BHT, quercetin, (-)-epicatechin, and (+)-catechin had no effect on tocopherol-omega-hydroxylase activity in HepG2 cells. In order to confirm the inhibition of gamma-T metabolism by sesame lignans in humans, sesame oil or corn oil muffins together with deuterium-labelled d6-alpha-Tand d2-gamma-Twere given to volunteers. Urine samples were collected for 72 h and analysed for deuterated and non-deuterated tocopherol metabolites. Consumption of sesame oil muffins significantly reduced the urinary excretion of d2-gamma-CEHC and total (sum of labelled and unlabelled) gamma-CEHC. Overall, the findings from these studies show that the tested dietary phenolic compounds increase vitamin E concentrations through different mechanisms and, thus, have the potential to improve vitamin E status without the use of vitamin E supplements.     

Comparison of extraction methods for quantifying vitamin E from animal tissues

Four extraction methods: (1) solvent (SOL), (2) ultrasound assisted solvent (UA), (3) saponification and solvent (SP), and (4) saponification and ultrasound assisted solvent (SP-UA), were used in sample preparation for quantifying vitamin E (tocopherols) in chicken liver and plasma samples. The extraction yields of SOL, UA, SP, and SP-UA methods obtained by adding delta-tocopherol as internal reference were 95%, 104%, 65%, and 62% for liver and 98%, 103%, 97%, and 94% for plasma, respectively. The methods with saponification significantly affected the stabilities of tocopherols in liver samples. The measured values of alpha- and gamma-tocopherols using the solvent only extraction (SOL) method were much lower than that using any of the other extraction methods. This indicated that less of the tocopherols in those samples were in a form that could be extracted directly by solvent. The measured value of alpha-tocopherol in the liver sample using the ultrasound assisted solvent (UA) method was 1.5-2.5 times of that obtained from the saponification and solvent (SP) method. The differences in measured values of tocopherols in the plasma samples by using the two methods were not significant. However, the measured value of the saponification and ultrasound assisted solvent (SP-UA) method was lower than either the saponification and solvent (SP) or the ultrasound assisted solvent (UA) method. Also, the reproducibility of the ultrasound assisted solvent (UA) method was greater than any of the saponification methods. Compared with the traditional saponification method, the ultrasound assisted solvent method could effectively extract tocopherols from sample matrix without any chemical degradation reactions, especially for complex animal tissue such as liver.     

Biotechnological production and application of vitamin E: current state and prospects

Tocochromanols (tocopherols and tocotrienols) are important lipophilic antioxidants for animals and humans. Their biological activity is expressed as vitamin E activity. This article describes the current need for vitamin E production, and compares different strategies to engineer the vitamin E content in photosynthetic bacteria and plants, with a focus on oilseed as target tissues. The current status of biotechnological advances in tocochromanol pathway engineering is summarized, and current limitations in our understanding of the tocochromanol biosynthetic pathway are discussed.     

Canola,corn, and vegetable oils as alternatives for wheat germ oil in fruit fly larval diets

Four wheat germ oil alternatives (corn oil, vegetable oil, canola oil with 10% vitamin E, and canola oil with 20% vitamin E), purchased from a local supermarket in Hawaii, were added to a fruit fly liquid larval diet as a replacement for wheat germ oil in the rearing of fruit fly larvae. The oils were tested on three species of fruit flies in Hawaii, Ceratitis capitata (TSL strain), Bactrocera dorsalis, and Bactrocera cucurbitae. They were evaluated for their efficacy in replacing WGO, based on: pupal recovery (%), larval duration (d), pupal weight (mg), adult emergence (%), adult fliers (%), mating (%), egg production per female per day, egg hatch (%), and peak egging period (d). Diets with WGO and without any oil were used as controls. The objective of the study was to select the most cost effective alternative oils with the best performance to replace the currently used WGO, which is pricey and hard to find. The results showed that there was no significant difference in performance among the tested oils in B. cucurbitae and B. dorsalis as regards the above mentioned parameters. Lower mating rate was observed in B. cucurbitae from those reared in vegetable oil and canola oil (10% vitamin E) diet. Lower egg production and egg hatch were obtained with B. dorsalis whose larvae were reared in vegetable and canola oil (both 10% and 20% vitamin E). Vegetable oil diet seemed to reduce pupal weight, shorten larval duration, and increase pupal recovery of C. capitata. The results suggest that WGO can be substituted with corn oil, vegetable oil, or canola oils for B. cucurbitae, while corn oil is a better alternative for B. dorsalis, and vegetable oil is best for C. capitata.     

Genotype‐dependent regulation of vitamin E biosynthesis in olive fruits as revealed through metabolic and transcriptional profiles

• Vitamin E is a general term used to describe a group of eight lipophilic compounds known as tocochromanols. These vitamin E variants are chemically categorised into two classes formed by α‐, β‐, γ‐ and δ‐ tocopherols and tocotrienols isoforms, respectively.
• The present study describes the concurrent regulation of genes and metabolites orchestrating vitamin E biosynthesis in olive drupes of five distinctive Greek olive cultivars. A combination of analytical, biochemical and molecular approaches was employed in order to carry out comparative analyses, including real‐time RT‐qPCR for gene expression levels and HPLC analysis of metabolite content.
• Findings indicated that tocochromanol levels and composition, oil content, gene expression levels as well as total antioxidant activity were highly dependent on cultivar and, to a lesser extent, on fruit developmental stage. Specifically, cultivars ‘Kalokairida’ and ‘Lianolia Kerkyras’ demonstrated the highest vitamin E content. The latter possessed high tocochromanol content combined with highest overall antioxidant activity in all developmental stages, concomitant with the up‐regulation expression profile of HPPD.
• The genotypic imprint versus the temporal contribution to vitamin E levels, as well as the potential link to lipid peroxidation amelioration, are discussed.

     

Wheat germ oil and its effects on a liquid larval rearing diet for oriental fruit flies (Diptera: Tephritidae)

Wheat germ oil was added to a larval liquid diet for rearing Bactrocera dorsalis (Hendel) (Diptera: Tephritidae) to optimize fruit fly quality. Effects of various concentrations of wheat germ oil at 0.04, 0.07, 0.15, 0.30, and 0.66% and their possible mode of action were evaluated. Results suggest that addition of wheat germ oil does not affect pupal weight, larval developmental period, adult emergence, mating ability, or peak time for egg production. But there was a significant increase in pupal recovery, percentage of adult fliers, egg production, or egg hatch for larvae fed the diet with wheat germ oil compared with those reared on the liquid diet without wheat germ oil. The increase in egg hatch and fliers was dose dependent. Therefore, addition of wheat germ oil to fruit fly rearing diet is a novel way to improve fruit fly quality, especially in egg hatch, fliers, egg production, and pupal recovery.     

Quality and lipid composition of spermatozoa in rabbits fed DHA and vitamin E rich diets

The effects of fish oil (FO) and vitamin E (vE) dietary supplementation on semen quality, sperm susceptibility to lipid peroxidation, tocopherols content and fatty acid profiles were studied in rabbits. Fifty-two rabbit bucks randomly divided in four groups received a control diet and enriched diets containing either FO (1.5%, w/w), vE (200 mg/kg) or both. Semen volume, concentration, motility and viability were analysed at various time-points and the lipid composition was assessed on sperm cells. The phospholipid fatty acid profile was determined: n-6 PUFA were the major fatty acids found, with a proportion of 42%, whereas the n-3 PUFA accounted for nearly 1%, mainly represented by C22:6n-3 (docosahexaenoic acid, DHA). FO supplementation produced a seven-fold increase in the content of DHA in sperm phospholipids and a comprehensive rearrangement of the phospholipid fatty acid composition, while an unexpected negative effect of feeding high level of vE on the proportion of total PUFA was found. Despite the remarkable changes observed in sperm lipid composition, semen quality parameters were not affected by the dietary treatments and the interaction between the two dietary supplements had a significant effect only on sperm concentration. An increase in semen production by ageing and a concomitant rise in sperm susceptibility to in vitro peroxidation was found. α- and δ-tocopherol, present in rabbit sperm in similar amount, were not affected by dietary treatment. δ-tocopherol content had a significant linear negative regression with age and showed a significant negative correlation with the susceptibility to peroxidation values.     

Production of tocopherols by cell culture of safflower

A new method for the production of tocopherols by safflower (Carthamus tinctorius) cell culture has been developed. The main tocopherol produced is α-tocopherol which has the strongest vitamin E activity among tocopherol analogues. In a time course experiment tocopherol production showed a secondary metabolic pattern rather than a primary one. Better cell lines in terms of both growth rate and amounts of tocopherols produced were obtained by selection using various growth regulators and media additives. In addition, tocopherol production was effectively stimulated by administration of biosynthetic precursors. In particular, phytol increased the total tocopherol content by some 18-fold, i.e. 63.6 mg per 100 g dry weight, and the α-tocopherol content by some 11- fold i.e. 28.8 mg per 100 g dry weight.     

The ectopic expression of the wheat Puroindoline genes increase germ size and seed oil content in transgenic corn

Plant oil content and composition improvement is a major goal of plant breeding and biotechnology. The Puroindoline a and b (PINA and PINB) proteins together control whether wheat seeds are soft or hard textured and share a similar structure to that of plant non-specific lipid-transfer proteins. Here we transformed corn (Zea mays L.) with the wheat (Triticum aestivum L.) puroindoline genes (Pina and Pinb) to assess their effects upon seed oil content and quality. Pina and Pinb coding sequences were introduced into corn under the control of a corn Ubiquitin promoter. Three Pina/Pinb expression positive transgenic events were evaluated over two growing seasons. The results showed that Pin expression increased germ size significantly without negatively impacting seed size. Germ yield increased 33.8% while total seed oil content was increased by 25.23%. Seed oil content increases were primarily the result of increased germ size. This work indicates that higher oil content corn hybrids having increased food or feed value could be produced via puroindoline expression.     

Pilot scale production of functional foods using red palm olein: Antioxidant,vitamins’ stability and sensory quality during storage

The objective of this research work was to produce acceptable quality functional foods, namely, extruded snacks, digestive biscuits and pan bread, on a pilot scale, using vitamin E and β-carotene-rich red palm olein (RPOL) and red palm shortening (RPS). These products were evaluated for their chemical composition and sensory quality along with the antioxidants and vitamin contents during the six months of storage at room temperature (22 ± 1 °C). Extruded snacks and digestive biscuits prepared with RPOL and RPS were found to be good sources of these antioxidant vitamins. The average β-carotene content of the control and test snacks at the end of six months of storage ranged from 26.8 to 56.1 mg/kg fat, and from 430.9 to 468.9 mg/kg fat, respectively. The total vitamin E content in control and test snacks made in Plant No. 1 decreased after six months of storage from 786.1 to 704.4 mg/kg fat, and from 765.1 to 695.4 mg/kg fat, respectively. As expected, the total tocotrienol content was four to five times higher than the total tocopherols in control biscuits. The RPOL containing 600–750 ppm of carotenes (mainly α- and β-carotenes), 710–774 ppm of vitamin E, was found to be suitable for industrial application in producing acceptable quality pan bread, digestive biscuits and snacks. These functional foods contained significant amounts of β-carotene and total vitamin E, indicating the possibility of producing such foods rich in these two of the important antioxidant vitamins coming from a natural source. The research findings strongly indicate that good-quality pan bread, extruded snacks and digestive biscuits can successfully be produced to offer healthier eating choices to the consumers of this region, thereby promoting better health and productivity among the population.     

The first two decades of vitamin E.

This presentation reviews highlights of the first 20 years (1922-1942) of vitamin E. It begins with background information leading to identification of an antisterility factor for rats of both sexes and its acceptance into the vitamin family as vitamin E (1925). Research of the next 12 years revealed a multiplicity of deficiency manifestations: embryonic mortality, testis degeneration, encephalomalacia and exudative diathesis in the chick, and nutritional muscular dystrophy in avian and mammalian species. Toward the close of this period came the isolation of vitamin E from natural sources, determination of its empirical formula, and introduction of the designation alpha-tocopherol for vitamin E (1936). Within the next two years the structural formula of alpha-tocopherol was elucidated, its chemical synthesis accomplished, and its production from natural plant oils by molecular distillation was well established. The existence of other tocopherols with lesser degrees of biological activity became recognized. Also, the concurrent development of a chemical method for determining the vitamin E content of alpha-tocopherol in foods, body tissues and body fluids, which replaced the very laborious bioassay procedure, greatly facilitated later advances in knowledge of the distribution and nature of vitamin E.     

Biosynthesis,regulation and functions of tocochromanols in plants

Tocopherols and tocotrienols have been originally identified as essential nutrients in mammals based on their vitamin E activity. These lipid-soluble compounds are potent antioxidants that protect polyunsaturated fatty acids from lipid peroxidation. The biosynthesis of tocopherols and tocotrienols occurs exclusively in photosynthetic organisms. The biosynthetic precursors and the different pathway intermediates have been identified by biochemical studies and the different vitamin E biosynthetic genes (VTE genes) have been isolated in several plants and cyanobacteria. The characterization of transgenic plants overexpressing one or multiple VTE genes combined with the study of vitamin E deficient mutants allows from now on understanding the regulation and the function of tocopherols and tocotrienols in plants.     

Overlapping photoprotective function of vitamin E and carotenoids in Chlamydomonas

Tocopherols (vitamin E) and carotenoids are the two most abundant groups of lipid-soluble antioxidants in the chloroplast. Carotenoids are well known for their roles in protecting against photooxidative stress, whereas the photoprotective functions of tocopherols have only recently been examined experimentally. In addition, little is known about the functional overlap of carotenoids and tocopherols in vivo. To investigate this possible overlap, Chlamydomonas reinhardtii strains were engineered to overproduce tocopherols by chloroplast transformation with non-codon-optimized and codon-optimized versions of the homogentisate phytyltransferase vitamin E2 (VTE2) from Synechocystis and by nuclear transformation with VTE2 from C. reinhardtii, which resulted in 1.6-fold, 5-fold to 10-fold, and more than 10-fold increases in total tocopherol content, respectively. To test if tocopherol overproduction can compensate for carotenoid deficiency in terms of antioxidant function, the nuclear VTE2 gene from C. reinhardtii was overexpressed in the npq1 lor1 double mutant, which lacks zeaxanthin and lutein. Following transfer to high light, the npq1 lor1 strains that overaccumulated tocopherols showed increased resistance for up to 2 d and higher efficiency of photosystem II, and they were also much more resistant to other oxidative stresses. These results suggest an overlapping functions of tocopherols and carotenoids in protection against photooxidative stress.     

Effect of Dietary Fat Quality and Vitamin E on the Antioxidant Potential of Pigs

A randomized blocked factorial experiment was conducted with 90 young pigs. From 4 to 11 weeks of age the pigs were kept in individual pens and fed a selenium supplemented basal diet consisting mostly of propionic acid treated barley, soybean meal and dried skim milk, and containing < 0.5 mg vitamin E per kg. The treatment factors during this period were 3 dietary levels of added vitamin E (nil, 10 and 30 mg/kg) and a 6 % supplement of fresh or oxidized fat (2/3 lard and 1/3 herring oil). From 11 weeks of age until slaughter at 90 kg the pigs received the vitamin E supplements but no fat or dried skim milk. The basal diet for this later experimental period was based on untreated dry barley. Blood samples collected during the period of investigation were examined for vitamin E and for resistance against erythrocyte lipid peroxidation (ELP) in order to evaluate the antioxidant status. Analysis of variance and Student’s t-test on least squares means showed the ELP to be influenced independently by the vitamin E supplement and, during the fat feeding period, by the quality of the fat supplement, with the highest peroxidation resistance (low ELP) in the groups fed fresh fat and a high level of vitamin E. Blood vitamin E level was only influenced — positively — by the vitamin E supplement although variations in the feed vitamin E level below 10–15 mg vitamin E per kg did not result in corresponding variations in measurable blood vitamin E concentrations. In the same low range of vitamin E in the feed there was a statistically significant difference in ELP values between the different vitamin E treatment groups. No clinical manifestations of selenium-vitamin E deficiency were observed in the pigs. The ELP and the plasma vitamin E levels observed would seem to suggest that a total of 15 mg vitamin E per kg barley-based feed will not always be sufficient for growing pigs.