Anna Prescha

Flavonoid engineering of flax potentiate its biotechnological application

BackgroundFlavonoids are a group of secondary plant metabolites important for plant growth and development. They show also a protective effect against colon and breast cancer, diabetes, hypercholesterolemic atherosclerosis, lupus nephritis, and immune and inflammatory reactions. Thus, overproduction of these compounds in flax by genetic engineering method might potentiate biotechnological application of these plant products.ResultsFlax plants of third generation overexpressing key genes of flavonoid pathway cultivated in field were used as plant material throughout this study. The biochemical properties of seed, oil and seedcake extracts and fibre from natural and transgenic flax plants were compared. The data obtained suggests that the introduced genes were stably inherited and expressed through plant generations.Overproduction of flavonoid compounds resulted in increase of fatty acids accumulation in oil from transgenic seeds due to protection from oxidation offered during synthesis and seed maturation. The biochemical analysis of seedcake extracts from seeds of transgenic flax revealed significant increase in flavonoids (kaempferol), phenolic acids (coumaric, ferulic, synapic acids) and lignan content. Fibres, another product of flax plant showed increase in the level of catechine and acetylvanillone and decrease in phenolic acids upon flax modification.Biochemical analysis results were confirmed using IR spectroscopy. The integral intensities of IR bands have been used for identification of the component of phenylpropanoid pathway in oil, seedcake extract and fibre from control and transgenic flax. It was shown that levels of flavonoids, phenolic acids and lignans in oil and seedcake extract was higher in transgenic flax products compared to control. An FT-IR study of fibres confirmed the biochemical data and revealed that the arrangement of the cellulose polymer in the transgenic fibres differs from the control; in particular a significant decrease in the number of hydrogen bonds was detected.ConclusionsAll analysed products from generated transgenic plants were enriched with antioxidant compounds derived from phenylopropanoid pathway Thus the products provide valuable source of flavonoids, phenolic acids and lignan for biomedical application. The compounds composition and quantity from transgenic plants was confirmed by IR spectroscopy. Thus the infrared spectroscopy appeared to be suitable method for characterization of flax products.

Effect of cellulose, pectin and chromium(III) on lipid and carbohydrate metabolism in rats.

The effects of cellulose, pectin and chromium(III) on lipid and carbohydrate metabolism were investigated in rats. Eighty male Buffalo rats (n=10/group, 4 weeks old) were fed experimental diets for 6 weeks. The two control groups received a fiber free diet (FF) or a fiber free diet plus chromium (FF+Cr) (2.53 mg Cr/kg diet). The other groups were fed diets containing 5% of cellulose (CEL), 5% of pectin (PEC) or 2.5% of cellulose plus 2.5% of pectin (CEL+PEC) with or without chromium. The daily food intake and body weight of the rats were not affected by the experimental diets. Total cholesterol level in plasma was significantly lower (p≤0.05) in the PEC group than the rats fed the FF diet. Feeding of rats with the PEC+Cr diet resulted in a significantly higher concentration of plasma HDL cholesterol (p≤0.05) when compared with the CEL+Cr group. No statistically significant differences in the concentrations of plasma triglycerides (TG) and non-esterified fatty acids (NEFA) between the groups were observed. Rats fed the CEL+Cr diet had a significantly lower content of cholesterol and rats fed the CEL+Cr diet lower contents of cholesterol and TG in the liver (p≤0.05) when compared with other groups. The concentration of HbA1c was significantly lower (p≤0.05) in rats fed the CEL and CEL+Cr diets than in other groups. A significantly lower concentration of plasma glucose (p≤0.05) was observed in rats receiving the CEL+PEC diet in comparison with the FF group. A significant effect of fiber and chromium combination was shown only in the case of triglyceride content in the liver of rats (p≤0.05). In conclusion, our results suggest that a diet containing fiber (PEC) and chromium or their supplements may be beneficial for correcting some disturbances of lipid metabolism, and a diet containing cellulose or its supplements may be used to improve glycemic control.

Characteristics of rose hip (Rosa canina L.) cold-pressed oil and its oxidative stability studied by the differential scanning calorimetry method.

Two new commercially available high linolenic oils, pressed at low temperature from rose hip seeds, were characterised for their composition, quality and DPPH radical scavenging activity. The oxidative stability of oils was assessed using differential scanning calorimetry (DSC). Phytosterols, tocopherols and carotenoids contents were up to 6485.4; 1124.7; and 107.7 mg/kg, respectively. Phenolic compounds determined for the first time in rose hip oil totalled up to 783.55 μg/kg, with a predominant presence of p-coumaric acid methyl ester. Antiradical activity of the oils reached up to 3.00 mM/kg TEAC. The acid, peroxide and p-anisidine values as well as iron and copper contents indicated good quality of the oils. Relatively high protection against oxidative stress in the oils seemed to be a result of their high antioxidant capacity and the level of unsaturation of fatty acids.

Engineering flax plants to increase their antioxidant capacity and improve oil composition and stability.

The composition of polyunsaturated fatty acids in the tissues is very important to human health and strongly depends on dietary intake. Since flax seeds are the richest source of polyunsaturated acids, their consumption might be beneficial for human health. Unfortunately, they are highly susceptible to auto-oxidation, which generates toxic derivatives. The main goal of this study was the generation of genetically modified flax plants with increased antioxidant potential and stable and healthy oil production. Since among phenylpropanoid compounds those belonging to the flavonoid route have the lowest antioxidant capacity, the approach was to inhibit this route of the pathway, which might result in accumulation of other compounds more effective in antioxidation. The suppression of the chalcone synthase gene resulted in hydrolyzable tannin accumulation and thus increased antioxidant status of seeds of the transgenic plant. This was due to the partial redirecting of substrates for flavonoid biosynthesis to the other routes of the phenylpropanoid pathway. Consequently, transgenic plants produced more (20-45%) polyunsaturated fatty acids than the control and mainly α-linolenic acid. Thus, increasing the antioxidant potential of flax plants has benefits in terms of the yield of suitable oil for human dietary consumption.

IR and Raman studies of oil and seedcake extracts from natural and genetically modified flax seeds

Flax plant of the third generation (F3) overexpressing key genes of flavonoid pathway cultivated in field in 2008 season was used as the plant material throughout this study. The biochemical properties of seed, oil and seedcake extracts from natural and transgenic flax plants were compared. Overproduction of flavonoids (kaempferol), phenolic acids (coumaric, ferulic/synapic) and lignan-secoisolariciresinol diglucoside (SDG) in oil and extracts from transgenic seeds has been revealed providing a valuable source of these compounds for biotechnological application. The changes in fatty acids composition and increase in their stability against oxidation along three plant generations were also detected. The analysis of oil and seedcake extracts was performed using Raman and IR spectroscopy. The wavenumbers and integral intensities of Raman and IR bands were used to identify the components of phenylpropanoid pathway in oil and seedcake extracts from control and transgenic flax seeds. The spectroscopic data were compared to those obtained from biochemical analysis.

Iron Status and Dietary Iron Intake of Vegetarian Children from Poland

Background/Aim: In Poland, vegetarian diets are becoming more and more popular. The aim of this study was to examine the effect of iron intake on iron status in vegetarian children. Methods: Dietary iron intake, iron food sources, blood count, serum iron, ferritin level and total iron-binding capacity were estimated in two groups of children, namely vegetarians (n = 22) and omnivores (n = 18) of both sexes, aged from 2 to 18 years. Seven-day food records were used to assess their diet. Results: Dietary iron intake in vegetarians and omnivores was low (up to 65.0 and 60.1% of the recommended dietary allowance). A significantly higher intake of vitamin C was observed in vegetarians compared with omnivores (p = 0.019). The main sources of iron in vegetarians were cereal products, followed by vegetables and mushroom products, then fruit. The prevalence of iron deficiency (ID) was higher in the vegetarian group (p = 0.023). The serum ferritin level and mean corpuscular volume in the vegetarians were also lower than in the omnivores (p = 0.01 and p = 0.014, respectively). Conclusions: Children who follow a vegetarian diet may suffer from ID in spite of having a high vitamin C intake. This indicates the need to introduce dietary education and iron status monitoring.

Quantitative and qualitative analysis of lipids in genetically modified potato tubers with varying rates of 14-3-3 protein synthesis

In six transgenic lines of potatoes with the varying rates of 14-3-3 protein synthesis as well as in control cultivar Desiree the content and composition of the lipids extracted from the mature tubers from three years field trials (1998-2000) were analyzed. The transgenic lines J2 and J1 are both overexpressing gene encoding 14-3-3 protein. The J2 exhibited an overexpression of the protein 14-3-3 derived from pumpkin (Cucurbita pepo) cDNA and in J1 the 14-3-3 overexpression resulted from modifying of ADP-ribosylation factor synthesis. In the remaining lines, synthesis of the protein 14-3-3 was modified by the antisense technology. In tubers from 1998, the content of total lipids was within the range of 0.45-0.88% of tuber dry matter. The highest amount of fat was in tubers of line J2 (69% more than in the control). The content of lipids in tubers from subsequent years ranged from 0.36 to 0.63% of dry matter. Consistently the highest amount of fat was in tubers of line J2, however, the increase was very slight (8.6% more than in the control). The fractionation of lipids into polar and nonpolar fractions showed that all transgenic lines from field trials 1998 and 2000 contained more nonpolar lipids than the control (up to 270% in line J2). The percentage of nonpolar fractions in fats of tubers from all transgenes harvested in 1999 were similar, but they were higher than in tubers from the previous years, and they amounted to 44.4-49.1%. Chromatographic separation of methyl esters of fatty acids demonstrated that cis-alpha-linoleic acid was the main fatty acid present in potato tubers. This acid composed the biggest part of all lipids in G2 line. In the nonpolar fraction of lipids, palmitic acid followed by cis-alpha-linoleic acid showed the highest amounts.

The influence of modified 14-3-3 protein synthesis in potato plants on the nutritional value of the tubers

Abstract The recently created six transgenic potato genotypes with overexpressed or underexpressed P14-3-3a (29G) and P14-3-3c (20R) isoforms of 14-3-3 protein were field-trialled (1998–2001). The contents of protein, starch, reducing sugars, sucrose and lipids were determined in the transgenic and control tubers harvested from the field. The obtained results showed a significant increase in crude protein content in potatoes with repression of P14-3-3c isoform and in potatoes with blocked P14-3-3a synthesis in comparison to the control line. A stable increase in lipid content of potatoes with overexpression of 14-3-3 protein from Cucurbita pepo in the field trials was observed. The variability of the investigated genotypes, in respect to the nutritional components, was statistically analysed using discriminant function and cluster analyses. The dominant influence of the variability of the genotypes exerted significant differentiation of protein, lipid and starch contents. These components showed the greatest discriminant power in the variability of genotypes. These results confirm the suggestion that 14-3-3 protein co-ordinates primary metabolite synthesis in plants.

Natural phenolics greatly increase flax (Linum usitatissimum) oil stability.

BackgroundFlaxseed oil is characterized by high content of essential polyunsaturated fatty acids (PUFA) promoted as a human dietary supplement protecting against atherosclerosis. The disadvantage of the high PUFA content in flax oil is high susceptibility to oxidation, which can result in carcinogenic compound formation. Linola flax cultivar is characterized by high linoleic acid content in comparison to traditional flax cultivars rich in linolenic acid. The changes in fatty acid proportions increase oxidative stability of Linola oil and broaden its use as an edible oil for cooking. However one of investigated transgenic lines has high ALA content making it suitable as omega-3 source. Protection of PUFA oxidation is a critical factor in oil quality. The aim of this study was to investigate the impact of phenylpropanoid contents on the oil properties important during the whole technological process from seed storage to grinding and oil pressing, which may influence health benefits as well as shelf-life, and to establish guidelines for the selection of new cultivars.MethodsThe composition of oils was determined by chromatographic (GS-FID and LC-PDA-MS) methods. Antioxidant properties of secondary metabolites were analyzed by DPPH method. The stability of oils was investigated: a) during regular storage by measuring acid value peroxide value p-anisidine value malondialdehyde, conjugated dienes and trienes; b) by using accelerated rancidity tests by TBARS reaction; c) by thermoanalytical - differential scanning calorimetry (DSC).ResultsIn one approach, in order to increase oil stability, exogenous substances added are mainly lipid soluble antioxidants from the isoprenoid pathway, such as tocopherol and carotene. The other approach is based on transgenic plant generation that accumulates water soluble compounds. Increased accumulation of phenolic compounds in flax seeds was achieved by three different strategies that modify genes coding for enzymes from the phenylpropanoid pathway. The three types of transgenic flax had different phenylpropanoid profiles detected in oil, highly increasing its stability.ConclusionsWe found that hydrophilic phenylpropanoids more than lipophilic isoprenoid compounds determine oil stability however they can work synergistically. Among phenolics the caffeic acid was most effective in increasing oil stability.

Serum and whole blood Zn, Cu and Mn profiles and their relation to redox status in lung cancer patients

Disturbed redox status may be critical to lung cancerogenesis, however little research has been conducted on general changes in total redox status in lung cancer. Levels and activities of antioxidants, especially enzymatic ones, are related to trace element concentration. Trace element status is often disturbed in cancers, however no studies concerning the association between redox and trace element status have been performed for lung cancer. We hypothesized that disturbed redox status in lung cancer patients is partially determined by trace elements while their distribution amongst blood compartments may differ compared to healthy subjects. Blood samples from lung cancer patients (n=44) and control subjects (n=44) were collected to assess redox and trace element status. Serum and whole blood Cu and Mn levels were determined with GF-AAS, and Zn-with F-AAS. In serum the total antioxidant status (TAS) was determined with the commercial kit TAS (Randox, UK), total oxidant status (TOS) was determined based on the method developed by Erel and the oxidative stress index (OSI) was calculated. Total protein (T-Prot), albumin (Alb), uric acid (UA) and total bilirubin (T-Bil) concentrations were measured with an auto-analyser (Konelab 20i, Thermoscientific, USA), SOD and CAT activity - with commercially available kits (Cayman, USA). The level of TAS, T-Prot, Alb, T-Bil, the activity of SOD, the concentration of whole blood Mn as well as serum and whole blood Zn were lower while TOS, OSI, serum Cu levels and serum Cu:Zn ratios were higher in lung cancer patients compared to the control group. In the lung cancer group TAS correlated positively with Alb and UA, serum Zn and negatively with whole blood Mn. Additionally, SOD positively correlated with the whole blood Mn and Cu:Zn ratio, while CAT - negatively with the whole blood Cu:Zn ratio. In the lung cancer sub-group at clinical stage I-II, TOS additionally negatively correlated with whole blood Zn, and CAT negatively with serum Cu and Cu:Zn ratio. In advanced lung cancer, we found a positive correlation between TAS and serum Zn, and a negative one - with serum Cu:Zn ratio. We observed a similar correlation between endogenous non-enzymatic antioxidants and TAS in the control group, however considerably fewer correlations between trace elements and antioxidants were observed. This study supports the hypothesis that disturbed redox status in lung cancer patients is linked with alterations in trace element status regarding Zn, Mn and Cu. Moreover, the type of biological fluid influences both - alterations in the metal profile and relationships with redox status parameters.