Protective Effect of Urtica dioica Extract against Oxidative Stress in Human Skin Fibroblasts.

Urtica dioica is a species with well-established significance in folk medicine in many countries. It was utilized to support the treatment of arthritis, allergies, and urinary tract disorders; however, the substantial presence of antioxidants suggests that nettle extract could also have a positive impact on the skin. The objective of this study was to assess the impact of nettle extract on human skin fibroblasts subjected to oxidative stress. Various solvents were tested to prepare an extract rich in polyphenolic compounds with high antioxidant potential. The chemical composition was determined using ultra-high-performance liquid chromatography with mass spectrometry (UPLC-DAD-MS). H2O2 treatment was used to induce oxidative stress and cell viability, and the metabolism was evaluated through NR and MTT assays. Our study demonstrated that extraction with 80% ethanol, followed by the drying and re-dissolving of the extract in pure water, was more efficient than direct extraction with water. This yielded an extract rich in polyphenolic compounds, with chlorogenic acid and caffeoylmalic acid as the predominant compounds, averaging 64.9 and 114.4 µg/mL, respectively. The extract exhibited antioxidant properties in the DPPH and ABTS assays. Furthermore, it did not exhibit cytotoxicity and did not negatively affect cell metabolism. In addition, it effectively reduced ROS in the H2O2-stimulated cells, and at the highest concentration tested, the ROS levels returned to those of the untreated control. The extract also protected against H2O2-induced cytotoxicity. The cell viability was maintained at the level of the untreated control when the cells were pretreated with the extract before H2O2 exposure. These findings indicate that U. dioica extract is a valuable and safe additive in skincare products.

The Antimicrobial Properties of Poplar and Aspen-Poplar Propolises and Their Active Components against Selected Microorganisms, including Helicobacter pylori.

There is a noticeable interest in alternative therapies where the outcome is the eradication of the Gram-negative bacterium, Helicobacter pylori (H. pylori), for the purpose of treating many stomach diseases (chronic gastritis and peptic ulcers) and preventing stomach cancer. It is especially urgent because the mentioned pathogen infects over 50% of the world's population. Recent studies have shown the potential of natural products, such as medicinal plant and bee products, on the inhibition of H. pylori growth. Propolis is such a bee product, with known antimicrobial activities. The main scope of the study is the determination of the antimicrobial activity of ethanolic extracts from 11 propolis samples (mostly from Poland, Ukraine, Kazakhstan, and Greece) against H. pylori, as well as selected bacterial and yeast species. The most effective against H. pylori was the propolis from Ukraine, with an MIC = 0.02 mg/mL while the rest of samples (except one) had an MIC = 0.03 mg/mL. Moreover, significant antimicrobial activity against Gram+ bacteria (with an MIC of 0.02-2.50 mg/mL) and three yeasts (with an MIC of 0.04-0.63 mg/mL) was also observed. A phytochemical analysis (polyphenolic profile) of the propolis samples, by ultra-high-performance liquid chromatography-diode array detector-mass spectrometry (UPLC-DAD-MS), was performed. An evaluation of the impact of the propolis components on antimicrobial activity, consisting of statistical analyses (principal component analysis (PCA) and hierarchical fuzzy clustering), was then performed. It was observed that the chemical composition characteristics of the poplar propolis correlated with higher antibacterial activity, while that of the poplar and aspen propolis correlated with weaker antibacterial activity. To summarize the activity in vitro, all tested propolis samples indicate that they can be regarded as useful and potent factors in antimicrobial therapies, especially against H. pylori.

Atherosclerosis Development and Aortic Contractility in Hypercholesterolemic Rabbits Supplemented with Two Different Flaxseed Varieties.

Alpha-linolenic acid (ALA) is widely regarded as the main beneficial component of flax for the prevention of cardiovascular disease. We evaluated the effect of the transgenic flaxseed W86-which is rich in ALA-on the lipid profile, atherosclerosis progression, and vascular reactivity in hypercholesterolemic rabbits compared to the parental cultivar Linola with a very low ALA content. Rabbits were fed a basal diet (control) or a basal diet supplemented with 1% cholesterol, 1% cholesterol and 10% flaxseed W86, or 1% cholesterol and 10% Linola flaxseed. A high-cholesterol diet resulted in an elevated plasma cholesterol and triglyceride levels compared to the control animals. Aortic sections from rabbits fed Linola had lower deposits of foamy cells than those from rabbits fed W86. A potassium-induced and phenylephrine-induced contractile response was enhanced by a high-cholesterol diet and not influenced by the W86 or Linola flaxseed. Pretreatment of the aortic rings with nitro-L-arginine methyl ester resulted in a concentration-dependent tendency to increase the reaction amplitude in the control and high-cholesterol diet groups but not the flaxseed groups. Linola flaxseed with a low ALA content more effectively reduced the atherosclerosis progression compared with the W86 flaxseed with a high concentration of stable ALA. Aorta contractility studies suggested that flaxseed ameliorated an increased contractility in hypercholesterolemia but had little or no impact on NO synthesis in the vascular wall.

Use of Natural Components Derived from Oil Seed Plants for Treatment of Inflammatory Skin Diseases.

The incidence of inflammatory skin diseases is increasing, so the search for relevant therapeutics is of major concern. Plants are rich in phytochemicals which can alleviate many symptoms. In this review, we concentrate on compounds found in the seeds of widely cultivated plants, regularly used for oil production. The oils from these plants are often used to alleviate the symptoms of inflammatory diseases through synergetic action of unsaturated fatty acids and other phytochemicals most commonly derived from the terpenoid pathway. The knowledge of the chemical composition of oil seeds and the understanding of the mechanisms of action of single components should allow for a more tailored approach for the treatment for many diseases. In many cases, these seeds could serve as an efficient material for the isolation of pure phytochemicals. Here we present the content of phytochemicals, assumed to be responsible for healing properties of plant oils in a widely cultivated oil seed plants and review the proposed mechanism of action for fatty acids, selected mono-, sesqui-, di- and triterpenes, carotenoids, tocopherol and polyphenols.

A new genotype of flax (Linum usitatissimum L.) with decreased susceptibility to fat oxidation: consequences to hematological and biochemical profiles of blood indices.

BACKGROUND: Flaxseed is an alternative to marine products that provide the traditional dietary sources of ω-fatty acids. A new genotype of flax, W92, is rich in natural antioxidants as well as having a reduced content of α-linolenic acid and therefore shows decreased susceptibility to fat oxidation. The objective of this study was to evaluate the effect of a diet supplemented with W92 flaxseed on hematological and biochemical blood indices. RESULTS: A positive impact of diet with the addition of flaxseed was observed on erythrocyte indices, including red blood cell (RBC), hematocrit (HCT), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) values. There were no significant differences for white blood cell (WBC), total protein and glucose values. Aspartate aminotransferase and alanine aminotransferase estimations in serum were also carried out and no obvious toxicity to the liver was shown. Moreover, a lipid profile was performed in serum samples and a decrease in total cholesterol and low-density lipoprotein cholesterol (LDL-C), accompanied by an increase in high-density lipoprotein cholesterol (HDL-C), was observed in rabbits fed flaxseed diets. CONCLUSION: Based on the results obtained, it appeared that the inclusion of a new genetically modified type of flaxseed in the diet altered cholesterol metabolism and could reduce the possibility of cardiovascular diseases. Diet enrichment with W92 flaxseed may be a solution to the health issues that are a result of improper diet in humans and animals. © 2016 Society of Chemical Industry.

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

BACKGROUND: Flaxseed 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. METHODS: The 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). RESULTS: In 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. CONCLUSIONS: We 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.

Genetically modified flax expressing NAP-SsGT1 transgene: examination of anti-inflammatory action.

The aim of the work was to define the influence of dietary supplementation with GM (genetically modified) GT#4 flaxseed cake enriched in polyphenols on inflammation development in mice liver. Mice were given ad libitum isoprotein diets: (1) standard diet; (2) high-fat diet rich in lard, high-fat diet enriched with 30% of (3) isogenic flax Linola seed cake; and (4) GM GT#4 flaxseed cake; for 96 days. Administration of transgenic and isogenic seed cake lowered body weight gain, of transgenic to the standard diet level. Serum total antioxidant status was statistically significantly improved in GT#4 flaxseed cake group and did not differ from Linola. Serum thiobarbituric acid reactive substances, lipid profile and the liver concentration of pro-inflammatory cytokine tumor necrosis factor-α were ameliorated by GM and isogenic flaxseed cake consumption. The level of pro-inflammatory cytokine interferon-γ did not differ between mice obtaining GM GT#4 and non-GM flaxseed cakes. The C-reactive protein concentration was reduced in animals fed GT#4 flaxseed cake and did not differ from those fed non-GM flaxseed cake-based diet. Similarly, the liver structure of mice consuming diets enriched in flaxseed cake was improved. Dietetic enrichment with GM GT#4 and non-GM flaxseed cakes may be a promising solution for health problems resulting from improper diet.

Bactericidal activities of GM flax seedcake extract on pathogenic bacteria clinical strains.

BACKGROUND: The antibiotic resistance of pathogenic microorganisms is a worldwide problem. Each year several million people across the world acquire infections with bacteria that are antibiotic-resistant, which is costly in terms of human health. New antibiotics are extremely needed to overcome the current resistance problem. RESULTS: Transgenic flax plants overproducing compounds from phenylpropanoid pathway accumulate phenolic derivatives of potential antioxidative, and thus, antimicrobial activity. Alkali hydrolyzed seedcake extract containing coumaric acid, ferulic acid, caffeic acid, and lignan in high quantities was used as an assayed against pathogenic bacteria (commonly used model organisms and clinical strains). It was shown that the extract components had antibacterial activity, which might be useful as a prophylactic against bacterial infection. Bacteria topoisomerase II (gyrase) inhibition and genomic DNA disintegration are suggested to be the main reason for rendering antibacterial action. CONCLUSIONS: The data obtained strongly suggest that the seedcake extract preparation is a suitable candidate for antimicrobial action with a broad spectrum and partial selectivity. Such preparation can be applied in cases where there is a risk of multibacterial infection and excellent answer on global increase in multidrug resistance in pathogenic bacteria.

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.

Flavonoid engineering of flax potentiate its biotechnological application.

BACKGROUND: Flavonoids 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. RESULTS: Flax 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. CONCLUSIONS: All 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.

New dressing materials derived from transgenic flax products to treat long-standing venous ulcers--a pilot study.

A new flax dressing product was developed based on three components (fibers, oil emulsion, and seedcake extract) from genetically engineered flax plants that were obtained by plant transformation using three genes controlling the synthesis of antioxidative compounds from the phenylpropanoid pathway. Simultaneous flax explant transformation with three genes coding for chalcone synthase, chalcone isomerase, and dihydroflavonol reductase resulted in an accumulation of phenolic acids in the fibers, polyunsaturated fatty acids in the oil, and lignans in the seedcake. The fibers, oil, and seedcake from transgenic flax contained a broad spectrum of antioxidative compounds. They were tested for cytotoxicity, and none were found to have a negative effect on the growth and morphology of Balb/3T3 cells. In this preliminary report, we present pilot data on the effects of using linen dressing treatment on its own or in combination with oil emulsion and/or seedcake extract on chronic wound healing. After a 12-week study, we concluded that an application of a modified flax-dressing (linen) bandage might yield a more rapid rate of healing and reduce the wound exudes and wound size. In several cases, wound healing was completed during the period of investigation. Interestingly and importantly, the patients reported that the new bandage made from modified flax diminished the pain accompanying chronic venous ulceration. Further study is required to determine any definitive effects of flax bandage on wound healing. This is the first pilot study report suggesting the benefits of a flax-based dressing on wound healing.

Engineering Flax with the GT Family 1 Solanum sogarandinum Glycosyltransferase SsGT1 Confers Increased Resistance to Fusarium Infection.

The aim of this study was to engineer a flax with increased resistance to pathogens. The approach was based on the recent analysis of the Solanum sogarandinum -derived glycosyltransferase (UGT) protein, designated SsGT1 (previously called 5UGT). On the basis of enzyme studies, the recombinant SsGT1 is a 7-O-glycosyltransferase, the natural substrates of which include both anthocyanidins and flavonols such as kaempferol and quercetin. Because flavonoids act as antioxidants and glycosylation increases the stability of flavonoids, it has been suggested that the accumulation of a higher quantity of flavonoid glycosides in transgenic plants might improve their resistance to pathogen infection. Flax overproducing SsGT1 showed higher resistance to Fusarium infection than wild-type plants, and this was correlated with a significant increase in the flavonoid glycoside content in the transgenic plants. Overproduction of glycosyltransferase in transgenic flax also resulted in proanthocyanin, lignan, phenolic acid, and unsaturated fatty acid accumulation in the seeds. The last is meaningful from a biotechnological point of view and might suggest the involvement of polyphenol glycosides in the protection of unsaturated fatty acids against oxidation and thus improve oil storage. It is thus suggested that introduction of SsGT1 is sufficient for engineering altered pathogen resistance in flax.

14-3-3 protein down-regulates key enzyme activities of nitrate and carbohydrate metabolism in potato plants.

The 14-3-3 protein is one of the best candidates for coordinating all plant metabolic pathways. To verify this suggestion transgenic potato plants with repression of one (J4 and J5 plants), two (G1 plants), and six (G3 plants) constitutive 14-3-3 protein isoforms as well as plants overexpressing the 14-3-3 protein were studied. Reduction in the 14-3-3 protein level in the J4 and J5 transformants, the G1 transformants, and the G3 transformants was close to 29, 41.5, 38, and 55%, respectively. In the case of the 14-3-3 overexpressing plants (J2), a 30% increase in protein content was detected. Changes in nitrate reductase (NR), sucrose phosphate synthase (SPS), and starch synthase (SS) activities in the transgenic plants perfectly reflect the overall 14-3-3 protein level. The highest increase in enzyme activities was observed for the G3 plants and the lowest for the J4 transformants. The same was detected for the measured metabolites. The highest increase in the protein, starch, and sucrose levels was detected in the tubers from the G3 transgenic plants. Because there was almost no change in the isoform ratio in the transgenic plants when compared to the control, it is suggested that it is the overall content of the 14-3-3 protein, rather than the content of particular isoforms, which plays a crucial role in the regulation of enzyme activities and thus in metabolite synthesis. The properties of the 14-3-3 overexpressing plants are very similar to those of the control ones, suggesting that the protein is in excess in the nontransformants and a further increase in its content is not recognized by cell metabolism. A considerable influence of the 14-3-3 protein level on potato plant metabolism was demonstrated. This effect was observed in key metabolic enzyme activities and metabolite content as well. A high variability between mean values, representing individual transgenes, with respect to nitrate reductase, sucrose phosphate synthase, and starch synthase activities in the examined genotypes was noted. These changes were closely correlated with metabolite levels, among them protein, starch, reducing sugars, and sucrose. The results obtained for the five types of transgenic potato plants in comparison with the control were statistically assessed using discriminate function and cluster analyses.

Pleiotropic effect of phenolic compounds content increases in transgenic flax plant.

The principal goal of this paper was to generate flax (Linum usitatissimum L.) plants with increased antioxidant properties. To accomplish this a vector containing a multigene construct was prepared, and transgenic plants overexpressing essential flavonoid biosynthesis pathway enzymes were generated and analyzed. The simultaneous expression of genes encoding chalcone synthase (CHS), chalcone isomerase (CHI), and dihydroflavonol reductase (DFR) resulted in a significant increase of flax antioxidant capacity. To investigate the determinants of higher antioxidant properties of transgenic plants, the phenolic acids and lignans compound contents were measured. In both green part and seed extracts from transgenic plants, the phenolic acids level was increased when compared to the control. The calculated correlation coefficient between phenolic acids content and antioxidant capacity (0.82 and 0.70 for green part and flaxseed, respectively) perfectly reflects their strong relationship. The increase in yield of transgenic plants and their higher resistance to Fusarium culmorum and Fusarium oxysporum when compared to the control plants was a characteristic feature. It was assessed a very high correlation (correlation coefficient = 0.9) between phenolic acids level in flaxseed extract and resistance to F. culmorum. The flowering date of transgenic plants was approximately 3 weeks earlier than that of the control plants. Interestingly, a significant increase in monounsaturated fatty acids and a slight increase in lignans content accompanied the increase in antioxidant properties of flaxseeds.

ADP ribosylation factor regulates metabolism and antioxidant capacity of transgenic potato tubers.

In our recent studies we have evidenced that repression of ADP-ribosylation factor (ARF) in potato plants results in 14-3-3 gene activation. The significant alteration in plant phenotype and in carbohydrate content clearly indicates that there may also be changes in other metabolite syntheses. In this paper we present the data on contents of compounds, occurring in transgenic potato tubers from field trial, known to be important for the human diet. We also determine which of the ARF-antisense plant features resulted from ARF repression. This determination was accomplished by the analysis of ARF-antisense plants transformed with cDNA encoding 14-3-3 protein in reverse orientation. The sucrose accumulation and the decrease in glycoalkaloids level were found to be characteristic features of all transgenic plants. The increase in antioxidant capacity of transgenic potato tubers should also be pointed out. The analysis of fat from modified potato tubers revealed a nutritionally valuable composition of fatty acids, including the significant increase of linoleic acid level.