The biomedical potential of genetically modified flax seeds overexpressing the glucosyltransferase gene
© Czemplik et al.; licensee BioMed Central Ltd. 2012
Received: 12 July 2012
Accepted: 7 December 2012
Published: 10 December 2012
Flax (Linum usitatissimum) is a potential source of many bioactive components that can be found in its oil and fibers, but also in the seedcake, which is rich in antioxidants. To increase the levels of medically beneficial compounds, a genetically modified flax type (named GT) with an elevated level of phenylopropanoids and their glycoside derivatives was generated. In this study, we investigated the influence of GT seedcake extract preparations on human fibroblast proliferation and migration, and looked at the effect on a human skin model. Moreover, we verified its activity against bacteria of clinical relevance.
The GT flax used in this study is characterized by overexpression of the glucosyltransferase gene derived from Solanum sogarandinum. Five GT seedcake preparations were generated. Their composition was assessed using ultra pressure liquid chromatography and confirmed using the UPLC-QTOF method. For the in vitro evaluation, the influence of the GT seedcake preparations on normal human dermal fibroblast proliferation was assessed using the MTT test and the wound scratch assay. A human skin model was used to evaluate the potential for skin irritation. To assess the antimicrobial properties of GT preparations, the percentage of inhibition of bacterial growth was calculated.
The GT seedcake extract had elevated levels of phenylopropanoid compounds in comparison to the control, non-transformed plants. Significant increases in the content of ferulic acid, p-coumaric acid and caffeic acid, and their glucoside derivatives, kaempferol, quercitin and secoisolariciresinol diglucoside (SDG) were observed in the seeds of the modified plants. The GT seedcake preparations were shown to promote the proliferation of normal human dermal fibroblasts and the migration of fibroblasts in the wound scratch assay. The superior effect of GT seedcake extract on fibroblast migration was observed after a 24-hour treatment. The skin irritation test indicated that GT seedcake preparations have no harmful effect on human skin. Moreover, GT seedcake preparations exhibited inhibitory properties toward two bacterial strains: Staphylococcus aureus and Escherichia coli.
We suggest that preparations derived from the new GT flax are an effective source of phenylopropanoids and that their glycoside derivatives and might be promising natural products with both healing and bacteriostatic effects. This flax-derived product is a good candidate for application in the repair and regeneration of human skin and might also be an alternative to antibiotic therapy for infected wounds.
KeywordsFlax Seedcake Phenylpropanoids Fibroblasts
Wound healing is a complex process that involves three main overlapping phases: inflammation, proliferation and tissue remodeling . It has been shown that many growth factors, cytokines and proteases are crucial for tissue repair after damage. The involvement of fibroblasts and keratinocytes is necessary to achieve total wound closure. Both types of cell migrate and undergo differentiation to restore the skin barrier . Adverse effects, such as possible pathogenesis, infections or ineffective healing, may significantly delay the process of wound closure. Therefore, the search for alternative compounds that improve wound healing is of great interest.
Recently, numerous research groups have reported on the traditional use of plants for wound healing. Annan and Houghton showed the significant effects on the growth of human dermal fibroblasts of extracts from Gossypium arboreum and Ficus asperifolia, and also reported on their protective effects on these cells against oxidative damage . The ethanol root extract of Ixora coccinea improved wound contraction and exhibited antibacterial activity . Increased proliferation and migration of keratinocites were observed in an in vitro study using aqueous extracts from the leaves of Chromolaena odorata. Phenolic compounds, i.e. flavonoids and phenolic acids, are known to regulate the normal human dermal fibroblast genes involved in antioxidant defense, the inflammatory response and cell renewal .
Recent research has focused on identifying the potential plant-derived agents that influence wound healing. The biological activity of plant extracts has been known for many years, as plants produce a wide range of phytochemicals. One source of such bioactive compounds is flax (Linum usitatissimum), which is widely distributed in Mediterranean and temperate climate zone. Flax seeds are known to play an important role in the food industry and health care. The known beneficial properties of flax are mainly associated with its oil, but a great amount of bioactive phytochemicals remains in the seedcake (the leftovers of the flax seeds after oil extraction). Flax seedcake contains phenolic acids, flavonoids, and other phenylopropanoids that are known to possess a wide range of biological activities and thus have a beneficial influence on human health . The valuable effects of phenylopropanoids are mainly due to their antioxidant properties. They prevent cardiovascular diseases, arteriosclerosis, cancers, inflammations and diabetes [8–10]. The seedcake is also a rich source of secoisolariciresinol diglucoside (SDG), the precursor of lignans, known to inhibit cancer cell proliferation and growth . SDG also possesses anti-bacterial, anti-fungal and anti-viral properties .
The beneficial features of flax products could clearly be enhanced if the secondary metabolite accumulation in the plant organs could be increased. In an attempt to increase the level of phenylpropanoid compounds in flax, a genetically modified flax type (named GT) with an elevated level of phenylopropanoids and their glycoside derivatives was generated. GT overexpresses the glucosyltransferase gene derived from Solanum sogarandinum in its seeds. Glycosyltransferases are enzymes that transfer activated sugar donors to other metabolites. In plants, phenylopropanoids can be acceptors of sugar moieties and are often converted to their glycoconjugates, which are then accumulated and compartmentalized in vacuoles . Glycosylation of phytochemicals is known to alter their regulatory properties by enhancing water solubility and increasing stability. Therefore, the performed modification aimed to increase the amount and stability of phenylopropanoids in flax seeds. In this study, we estimated the effect of GT flax seedcake extracts on the growth and migration of human dermal fibroblasts, and we determined the potential for skin irritation using a human skin model.
Flax seeds (cv. Linola 947) were obtained from the Flax and Hemp Collection of the Institute of Natural Fibers, Poland. Transgenic plant construction and selection was performed as previously described by Lorenc-Kukuła et al. . The GT plants (previously called UGT plants) overexpress SsGT1 (EMBL/GenBank accession no. AY033489) from Solanum sogarandinum under a seed- specific napin promoter (EMBL/GenBank, accession no. J02798) and the OSC terminator. In this study, the third generation of GT transgenic plants line #4 (GT4) was grown in a field, and the seeds were harvested 3 months after sowing, in parallel, the control plants (cv. Linola 947, obtained from the Flax and Hemp Collection of the Institute of Natural Fibers, Poland) were grown in the same location (Lower Silesia, Poland) in the same year. GT4 and Linola seeds were pressed to obtain oil on an industrial warm gear oil press (Oil PressDD85G – IBG Monoforts Oekotec GmbH& Co). The GT4 and Linola seedcakes were collected and used for further experiments.
Identification and quantification of phenylpropanoids in flax seedcake extracts
A 0.25 g sample of GT4 flax seedcakes was extracted three times with 1.5 mL of 80% methanol (v/v) for 10 min at 80°C. Prior to extraction, the seedcakes were defatted with hot hexane. The extract was centrifuged and evaporated at 40°C under a vacuum and then resuspended in water and subjected to alkaline hydrolysis (1 mL, 0.3 M aqueous sodium hydroxide) for 2 days at room temperature followed by neutralization using 2 M hydrochloric acid . The extract was analyzed on a Waters Acquity UPLC system with a 2996 PDA detector, using an Acquity UPLC column BEH C18, 2.1100 mm, 1.7 μm. The mobile phase was A = acetonitrile and B = 20 mM ammonium formate, pH 3, in a gradient flow: 1 min, 10%/90% A/B, 2–6 min gradient to 40%/60% A/B, and 7 min gradient from 40% to 100% A with a 0.4 mL/min flow rate. The compounds were measured at 280 and 320 nm.
The identity of the components was confirmed by LC-MS analysis on a Waters Aquity UPLC-QTOF system using BEH C18, 2.1 150 mm, 1.7 μm The mobile phase was A = acetonitrile and B = 0.1% formic acid, in a gradient flow: 1 min, 10%/90% A/B, 2–10 min gradient to 80%/20% A/B, and 12 min gradient from 80% to 100% A with a 0.4 mL/min flow rate. The MS spectra were recorded in ESI positive mode for 13 min in 50–800 Da range. The parameters were: nitrogen flow 800 L/h, source temperature 70°C, desolvation temperature cone 400°C, capillary voltage 3.50 V, sampling cone 30 V, cone voltage ramp 40–60 V, scan time 0.2 sec. The obtained spectra were compared to those of known standards and data in the literature.
The components of GT4 seedcake preparations
Biochemical composition of GT4 seedcakes preparations used for cell in vitro studies
Ferulic acid glucoside
p-coumaric acid glucoside
Caffeic acid glucoside
Antioxidant activity of GT4 preparations
Radical scavenging activity of the GT4 seedcake preparations and pure compounds was determined using the stable free radical 2,2’-diphenylpicrylhydrazyl (DPPH) method. A quantity of 40 μl of each test solution (GT4 preparation No. 2, 3 and 4, 1 mM ferulic acid, 1 mM p-coumaric acid, 1 mM quercetin, 1 mM rutin, 1 mM vitamin C) was mixed with 1000 μl of a freshly prepared DPPH-methanol solution (0,1 mM) and allowed to stand for 30 min at room temperature. The optical densities of the resulting solutions were read at 517 nm using a Cary 50 Conc UV/vis spectrometer.
Normal human dermal firbroblasts (NHDF) were obtained from Laboratory of Cell Pathology, Faculty of Biotechnology at Wrocław University, Poland. The NHDF cells were maintained at 37°C, 5% CO2 in Minimum Essential Medium Alpha (α-MEM, Institute of Immunology and Experimental Therapy, Polish Academy of Science, Poland) supplemented with 10% fetal bovine serum (FBS, Lonza, USA), 1% L-glutamine (Invitorgen, USA) and 1% antibiotic mixture (Invitorgen, USA). NHDF used in this study were between the third and seventh passages. 24 h prior to the treatment, the medium was replaced with α-MEM containing 0.5% FBS. The 0.5% FBS concentration is a maintenance dose needed for the production of healthy cells. It does not significantly stimulate proliferation of cells.
Cells were seeded in a 24-well plate at a concentration of 1 × 104 cells/mL, and after 24 h GT4 seedcake preparations (#1-#5) were added to the plate. Non-treated cells served as a control. To assess the effect of the GT4 seedcake extracts on the growth of the cells, microscopic observations were performed after 24 h and 48 h using a transmitted light phase contrast microscope equipped with × 10 and × 100 objectives (Axiovert 40 CFL, ZEISS).
Cell proliferation assay
Cells were seeded in a 24-well plate at concentration of 1 × 104 cells/mL, and after 24 h GT4 seedcake preparations (#1-#5) were added to the plate. Non-treated cells served as a control. To assess the proliferation potential, NHDF cells were incubated and assayed after 72 h. After this period of treatment, 50 μL of MTT stock solution (4 mg/mL) was added to each well to give a total reaction volume of 550 μL. After incubating for 4 h, the medium with MTT solution was removed from the plate. The formazan crystals in each well were dissolved in 500 μL of DMSO and incubated for 30 min with gentle shaking. The absorbance at 540 nm was read on an Asys UVM340 Microplate Reader (Biochrom, UK). The MTT assay was performed in triplicate. The results were presented in % as a reference to the control (100%).
In vitro wound scratch assay
Wound-healing properties were evaluated using the in vitro scratch assay , which measures the expansion of a cell population on surfaces. NHDF cells were seeded in a 24-well plate at concentration of 1 × 104 cells/mL, and maintained to nearly confluent cell monolayers. Next, a linear wound was generated in the monolayer with a sterile 100-μL plastic pipette tip. Any cellular debris was removed by washing with phosphate buffer saline (PBS). α-MEM containing 0.5% FBS, 1% L-glutamine (Invitrogen, USA) and 1% antibiotic mixture (Invitrogen, USA) was added to the plate. Then, the GT4 seedcake preparations (#1-#4) were added. Non-treated cells served as a control. To estimate the relative migration of NHDF cells, three representative images of the scratched areas from each well were photographed using a transmitted light phase contrast microscope equipped with × 10 and × 100 objectives (Axiovert 40 CFL, ZEISS). Wound closure was determined as the difference in expansion area at 0, 24 and 48 h. The experiments were performed at least in triplicate. The data were analyzed using TScratch software.
Skin irritation test
An in vitro skin irritation test was performed according to the MTT Effective Time-50 (ET-50) protocol developed at MatTek Corporation with the use of the EpiDerm skin irritation test. This test allows the assessment of skin irritation due to cosmetic ingredients and ready-made products. 100 μL of three preparations of GT4 seedcake extracts (#2, #4 and the concentrated, non-diluted preparation #36) were put on the surface of the epidermis model. After an incubation period of 2, 5 or 18 h, cell viability was assessed using the MTT colorimetric test (described above). Skin irritation potential is predicted if the remaining relative cell viability is below 50%. The experiment was performed in duplicate.
Staphylococcus aureus strain ATCC 6538 and Escherichia coli strain ATCC 10536 were used as indicators for antimicrobial testing and were obtained from the in-house culture collection of the Microbiology Department at the University of Wrocław, Poland. Bacteria were grown at 37°C in Luria-Bertani (LB) medium under shaking conditions. Diluted (100-fold in LB) overnight cultures of all of the tested bacteria strains (150 μL) were incubated in 96-well plates at 37°C, shaking at 600 rpm with a previously prepared seedcake extract of GT4 flax. The volumes contained the standardized proportions of 0.5 to 25 mg/ml SDG. OD600 was monitored at 4, 6 and 12 h in an Asys UVM340 Microplate Reader (Biochrom, UK).
Results and discussion
Identification and quantification of phenylopropanoids in seedcake extracts of GT flax
UPLC-QTOF identification of phenylpropanoids of seedcake extracts
PDA max (nm)
Caffeic acid glucoside
240, 290 (A max), 317 (sh)
163[M-Glu-H2O + H]+
181[M-Glu + H] +,
343 [M-H20 + H] +
p-coumaric acid glucoside
231, 296 (A max)
147[M-Glu-H2O + H]+
165[M-Glu + H] +,
327 [M-H20 + H] +
Ferulic acid glucoside
237, 291 (A max), 316 (sh)
177[M-Glu-H2O + H]+
195[M-Glu + H] +,
357 [M-H20 + H] +
240, 297 (sh), 322 (A max)
163[M-H2O + H] +,
181[M + H] +
230, 300 (sh), 309(A max)
147[M-H2O + H]+,
165[M + H] +
Secoisolariciresinol diglucoside (SDG)
233, 281 (A max)
327[M-2Glu + H]+,
525[M-Glu + H20 + H]+,
687[M + H]+
246, 297 (sh), 323 (A max)
177[M-H2O + H] +,
195[M + H] +
Composition of the seedcake extracts of transgenic GT4 and control (Linola) flax
Secoisolariciresinol diglucoside (SDG)
19095 ± 118
36880 ± 13
51 ± 2.6
87 ± 3.6
p-coumaric acid glucoside
12621.57 ± 23
19702.4 ± 613
805.1 ± 57
1175 ± 62
Ferulic acid glucoside
87570 ± 34
179386 ± 942
28.2 ± 2.0
16.6 ± 5.5
Caffeic acid glucoside
64248 ± 37
90636 ± 76
Moreover the previous preliminary research on the cell cycle of fibroblasts treated with seedcake extracts of Linola seeds and GT4 seeds indicated that the percentage of proliferating fibroblast was higher for GT4 preparation treated cells than for Linola preparation treated cells (data not shown). For these reason GT4 modified fax has been chosen as an object for further studies, presented in this manuscript.
Effects of GT4 seedcake preparations on the growth and proliferation of normal human dermal fibroblasts
Due to the reported antioxidant properties of flax seed constituents, it was suggested that GT4 seedcakes might be a great source of health-promoting compounds. To investigate their effect on human dermal fibroblasts and a human skin model, five different dilutions of GT4 seedcake extract were prepared (#1-#5). The detailed data for the phenylopropanoid contents in the GT4 seedcake extracts used for our in vitro cell culture experiments are presented in Table 1.
During wound healing, the balance between oxidative and antioxidative processes is disturbed, which results in excessive free radical generation. This oxidative stress is a crucial factor in wound progression and pathogenesis . It promotes the damage of many cellular components and mechanisms and causes fibroblast apoptosis . In chronic wounds, the endogenous antioxidants are not sufficient for free radical neutralization, so providing additional antioxidants might restore oxidative homeostasis .
It is suggested that plant-derived compounds of the phenylopropanoid pathway have a positive influence on wound healing. There are some reports confirming the beneficial influence of phenolic plant extracts on fibroblasts. Kim et al. showed the enhanced proliferation of human skin fibroblasts treated with Ginkgo biloba extract. This proliferative effect is due to the action of phenylopropanoids . It was also reported that a leaf extract of Wedelia trilobata displayed a stimulatory effect on fibroblasts and promoted the synthesis of collagen . Therefore, it was suggested that the extract from seedcakes of modified flax with elevated levels of phenylpropanoids might improve the proliferation and migration of human fibroblasts.
Indeed, our study revealed that GT4 seedcake preparations enhanced normal human dermal fibroblast proliferation by 30%. Such a rate of increase is not spectacular, but a significant increase in the proliferation potential of fibroblasts is not favorable during wound healing, as it promotes overgrowth of connective tissue and scar formation.
On the other hand the relationship between fibroblasts growth in in vitro cultures and in vivo wound closure are not elucidated yet, so the in vivo effect to of GT4 seedcake preparation extract might be different and eventually less significant. Moreover, the growth and mobility of fibroblasts depends on the presence of the extracellular matrix, so these effects are still to be elucidated in in vivo experiments. Of all the preparations tested, the one with the highest phenylpropanoid content (#5) exhibited negative effects on NHDF cells, which was observed in the microscopic study and proliferation test. It is estimated that such a high concentration of phenylpropanoids might result in the predominance of pro-oxidative processes over antioxidative processes, probably due to the reduction of chelated Fe3+ to Fe2+ and the subsequent radical generation by metal-induced oxidation . This might partially explain the toxic effect of preparation #5 to cells. Therefore, preparation #5 was excluded from the following experiment.
Migration of GT seedcake-treated fibroblasts in wound scratch assay
One of the crucial processes for wound closure is the migration of fibroblasts. They migrate to the wound, form granulation tissue, and create the basis for the wound closure . The migration potential of NHDF cells treated with GT4 seedcake extracts was assessed in the scratch assay, a method that mimics the migration of cells in vivo and is commonly used to study cell migration in vitro. For the experiment, we chose GT4 seedcake preparations #1, #2, #3 and #4, which exhibited no negative effect on fibroblast growth or proliferation. The observations of fibroblast migration were performed using light microscopy and the migration was determined as the expansion of a cell population on surfaces after 24 and 48 h.
The presented results suggest the GT4 seedcake preparations are effective in enhancing migration of NHDF cells, but only during the first 24 h. The phenylpropanoids in GT4 seedcake are thus thought to be responsible for the increase of NHDF cell migration, but their action is time-limited. It is also of note that the highly concentrated GT4 seedcake extract #4 was practically ineffective in the acceleration of wound closure. Preparation #1 slowed migration of NHDF cells suggesting that the lowest concentration of phenylpropanoids night not enhance the mobility of skin fibroblasts but enhance their growth. Migration is a complex process that involves coordinated polymerization, adhesion, actin polymerization, and movement to the membrane in the migration direction. To find out which phases are affected by GT4 seedcake preparations, further experiments must be performed. Fibroblast proliferation and migration are independent mechanisms, which might partially explain the different effects of GT4 seedcake preparations on these two processes. Similarly, the mechanism of influence on NHDF proliferation remains to be elucidated.
Evaluation of skin irritation caused by GT seedcake preparations
To better assess the application potential of GT4 seedcake preparations, the skin irritation test was performed with use of EpiDerm, a three-dimensional human skin model. This is a fully developed epidermis with a functional stratum corneum, i.e. with keratinocytes in various stages of differentiation. It is commonly used to assess the irritation potential of dermally applied substances. This test can be applied as an alternative for evaluating dermal irritation prior to clinical tests. The GT4 seedcake preparations were directly applied to the stratum corneum of this air-lifted, highly differentiated culture. The in vitro skin irritation evaluation was based on the MTT test and assessed 2, 5 and 18 h after application of the preparations.
Additionally, we tested the effect of non-diluted GT4 seedcake extract (#36) on the human skin model. The cell viability significantly decreased, which indicates the concentrated GT4 seedcake extract possesses properties that irritate the skin.
We suggest that high concentrations of phenylopropanoids in GT4 seedcake preparations are toxic to keratinocytes. Preparations #1-#4 appeared to be safe for dermatological applications, but caution must be taken if using highly concentrated extracts, since proliferation of fibroblasts can be affected and an irritation reaction can occur.
Antimicrobial properties of GT4 seedcake preparations
Wounds are exposed to the external environment and are thus prone to attack by microbes, which delay the wound-healing process. Therefore, the antimicrobial activity of GT4 seedcake extracts was assessed on strains of common pathogens of human skin. According to the literature, 27% of chronic wounds are infected with E. coli and 57.1% with S. aureus. Thus, preparations dedicated to improve the healing of chronic wounds should also be antibacterial agents.
Our findings demonstrate that genetic engineering can be used to improve the qualities of flax seeds. Overexpression of the glucosyltransferase gene in seeds increased the content of phenylopropanoids and their glucoside derivatives. Flax seedcakes rich in these compounds might be a promising source of products that are beneficial for human health. The positive influence on fibroblast proliferation and migration and the bacteriostatic effects suggest that GT4 seedcake preparations might be a good candidate for the repair and regeneration of human skin and an alternative to antibiotic therapy of infected wounds. Moreover, our results show that these preparations are safe for use on the skin. Such new application possibilities of flax and especially their biomedical relevance can contribute to the renewal of flax cultivation worldwide.
Flax type overexpressing glucosyltransferase gene derived from Solanum sogarandinum in flax seeds
Line # of flax type overexpressing glucosyltransferase gene derived from Solanum sogarandinum in flax seeds.
This study is supported by the grants NR 12-0009-06, NR 12017110 from the Ministry of Education and Sciences and UDA-POIG.01.04.00-22-022/11-00 from the EU Innovative Economy Programme.
- Kondo T, Ishida Y: Molecular pathology of wound healing. Forensic Sci Int. 2010, 203: 93-98. 10.1016/j.forsciint.2010.07.004.View ArticlePubMedGoogle Scholar
- Singer AJ, Clark RA: Cutaneous wound healing. N Engl J Med. 2007, 341: 738-746.Google Scholar
- Annan K, Houghtonb PJ: Antibacterial, antioxidant and fibroblast growth stimulation of aqueous extracts of Ficus asperifolia Miq. and Gossypium arboreum L., wound-healing plants of Ghana. J Ethnopharmacol. 2008, 119: 141-144. 10.1016/j.jep.2008.06.017.View ArticlePubMedGoogle Scholar
- Selvaraj N, Lakshmanan B, Mazumder PM, Karuppasamy M, Jena SS, Pattnaik AK: Evaluation of wound healing and antimicrobial potentials of Ixora coccinea root extract. Asian Pac J Trop Med. 2011, 4: 959-963. 10.1016/S1995-7645(11)60226-5.View ArticlePubMedGoogle Scholar
- Phan TT, Hughes MA, Cherry GW: Effects of an aqueous extract from the leaves of Chromolaena odorata (Eupolin) on the proliferation of human keratinocytes and on their migration in an in vitro model of reepithelialization. Wound Repair Regen. 2001, 9: 305-313. 10.1046/j.1524-475X.2001.00305.x.View ArticlePubMedGoogle Scholar
- Dudonné S, Poupard P, Coutière P, Woillez M, Richard T, Mérillon JM, Vitrac X: Phenolic composition and antioxidant properties of poplar bud (Populus nigra) extract: individual antioxidant contribution of phenolics and transcriptional effect on skin aging. J Agric Food Chem. 2011, 59: 4527-4536. 10.1021/jf104791t.View ArticlePubMedGoogle Scholar
- Korkina LG: Phenylpropanoids as naturally occurring antioxidants: from plant defense to human health. Cell and Molecular Biology. 2007, 53: 15-25.Google Scholar
- Halliwell B: Dietary polyphenols: good, bad, or indifferent for your health?. Cardiovasc Res. 2007, 73: 341-347. 10.1016/j.cardiores.2006.10.004.View ArticlePubMedGoogle Scholar
- Décordé K, Teissèdre PL, Auger C, Cristol JP, Rouanet JM: Phenolics from purple grape, apple, purple grape juice and apple juice prevent early atherosclerosis induced by an atherogenic diet in hamsters. Mol Nutr Food Res. 2008, 52: 400-407. 10.1002/mnfr.200700141.View ArticlePubMedGoogle Scholar
- Qu H, Madl RL, Takemoto DJ, Baybutt RC, Wang W: Lignans are involved in the antitumor activity of wheat bran in colon cancer SW480 cells. J Nutr. 2005, 135: 598-602.PubMedGoogle Scholar
- Wang L, Chen J, Thompson LU: The inhibitory effect of flaxseed on the growth and metastasis of estrogen receptor negative human breast cancer xenograftsis attributed to both its lignan and oil components. Int J Cancer. 2005, 116: 793-798. 10.1002/ijc.21067.View ArticlePubMedGoogle Scholar
- Rajesha J, Ranga Rao A, Madhusudhan B, Karunakumar M: Antibacterial Properties of Secoisolariciresinol Diglucoside Isolated from Indian Flaxseed Cultivars. Current Trends in Biotechnology and Pharmacy. 2010, 4: 551-560.Google Scholar
- Lim EK, Bowles DJ: A class of plant glycosyltransferases involved in cellular homeostasis. EMBO J. 2004, 23: 2915-2922. 10.1038/sj.emboj.7600295.View ArticlePubMedPubMed CentralGoogle Scholar
- Lorenc-Kukuła K, Żuk M, Kulma A, Czemplik M, Kostyn K, Skala J, Starzycki M, Szopa J: Engineering Flax with the GT Family 1 Solanum sogarandinum Glycosyltransferase SsGT1 confers increased resistance to Fusarium infection. J Agric Food Chem. 2009, 57: 6698-6705. 10.1021/jf900833k.View ArticlePubMedGoogle Scholar
- Johnsson P, Kamal-Eldin A, Lundgren LN, Aman P: HPLC method for analysis of secoisolariciresinol diglucoside in flaxseeds. J Agric Food Chem. 2000, 48: 5216-5219. 10.1021/jf0005871.View ArticlePubMedGoogle Scholar
- Gebäck T, Schulz MM, Koumoutsakos P, Detmar M: TScratch: a novel and simple software tool for automated analysis of monolayer wound healing assays. Biotechniques. 2009, 46: 265-274.PubMedGoogle Scholar
- Hu C, Yuan YV, Kitts DD: Antioxidant activities of the flaxseed lignan secoisolariciresinol diglucoside, its aglycone secoisolariciresinol and the mammalian lignans enterodiol and enterolactone in vitro. Food Chem Toxicol. 2007, 45: 2219-2227. 10.1016/j.fct.2007.05.017.View ArticlePubMedGoogle Scholar
- Kasote DM, Hegde MV, Deshmukh KK: Antioxidant activity of phenolic components from n-butanol fraction (PC-BF) of defatted flaxseed meal. Am J Food Technol. 2011, 6: 604-612.View ArticleGoogle Scholar
- Karamać M, Kosińska A, Pegg R: Comparison of radical-scavenging activities for selected phenolic acids. Pol J Food Nutr Sci. 2005, 55: 165-170.Google Scholar
- Brand-Williams W, Cuvelier ME, Berest C: Use of a free radical method to evaluate antioxidant activity. LWT- Food Sci Technol. 1995, 28: 25-30. 10.1016/S0023-6438(95)80008-5.View ArticleGoogle Scholar
- Clark R: Oxidative stress and ''senescent'' fibroblasts in non-healing wounds as potential therapeutic targets. J Invest Dermatol. 2008, 128: 2361-2364. 10.1038/jid.2008.257.View ArticlePubMedGoogle Scholar
- Wall IB, Moseley R, Baird DM, Kipling D, Giles P, Laffaian I, Price PE, Thomas DW, Stephens P: Fibroblast dysfunction is a key factor in the non-healing of chronic venous leg ulcers. J Invest Dermatol. 2008, 128: 2526-2540. 10.1038/jid.2008.114.View ArticlePubMedGoogle Scholar
- Bonnefoy M, Drai J, Kostka T: Antioxidants to slow aging, facts and perspectives. Presse Med. 2002, 31: 174-184.Google Scholar
- Kim SJ, Lim MH, Chun IK, Won YH: Effects of flavonoids of Ginkgo biloba on proliferation of human skin fibroblast. Skin Pharmacol. 1997, 10: 200-205. 10.1159/000211505.View ArticlePubMedGoogle Scholar
- Balekar N, Katkam NG, Nakpheng T, Jehtae K, Srichana T: Evaluation of the wound healing potential of Wedelia trilobata (L.) leaves. J Ethnopharmacol. 2012, 141: 817-824. 10.1016/j.jep.2012.03.019.View ArticlePubMedGoogle Scholar
- Stadler RH, Markovic J, Turesky RJ: In vitro anti- and pro-oxidative effects of natural polyphenols. Biol Trace Elem Res. 1995, 47: 299-305. 10.1007/BF02790130.View ArticlePubMedGoogle Scholar
- Skórkowska-Telichowska K, Bugajska-Prusak A, Pluciński P, Rybak Z, Szopa J: Fizjologia i patologia przewlekle niegojących się owrzodzeń oraz sposoby ich miejscowego leczenia w świetle współczesnej wiedzy medycznej. Dermatologia Praktyczna. 2009, 5: 15-28.Google Scholar
- Liang CC, Park AY, Guan JL: In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nat Protoc. 2007, 2: 329-333.View ArticlePubMedGoogle Scholar
- Dąbrowiecki S: Fizjologia i patofizjologia procesu gojenia ran. Polska Medycyna Paliatywna. 2003, 2: 290-291.Google Scholar
- Kosalec I, Pepeljnjak S, Bakmaz M, Vladimir-Knazevic S: Flavonoid analysis and antimicrobial activity of commercially available propolis products. Acta Pharmacol. 2005, 55: 423-430.Google Scholar
- Kim SY, Kang DH, Kim JK, Ha YG, Hwang JY, Kim T, Lee SH: Antimicrobial activity of plant extracts against Salmonella typhimurium, Escherichia coli O157:H7, and Listeria monocytogenes on fresh lettuce. J Food Sci. 2011, 76: 41-46.View ArticleGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6882/12/251/prepub
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.