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1.  Flavonoid engineering of flax potentiate its biotechnological application 
BMC Biotechnology  2011;11:10.
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.
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.
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.
PMCID: PMC3040132  PMID: 21276227
2.  Physical mapping and BAC-end sequence analysis provide initial insights into the flax (Linum usitatissimum L.) genome 
BMC Genomics  2011;12:217.
Flax (Linum usitatissimum L.) is an important source of oil rich in omega-3 fatty acids, which have proven health benefits and utility as an industrial raw material. Flax seeds also contain lignans which are associated with reducing the risk of certain types of cancer. Its bast fibres have broad industrial applications. However, genomic tools needed for molecular breeding were non existent. Hence a project, Total Utilization Flax GENomics (TUFGEN) was initiated. We report here the first genome-wide physical map of flax and the generation and analysis of BAC-end sequences (BES) from 43,776 clones, providing initial insights into the genome.
The physical map consists of 416 contigs spanning ~368 Mb, assembled from 32,025 fingerprints, representing roughly 54.5% to 99.4% of the estimated haploid genome (370-675 Mb). The N50 size of the contigs was estimated to be ~1,494 kb. The longest contig was ~5,562 kb comprising 437 clones. There were 96 contigs containing more than 100 clones. Approximately 54.6 Mb representing 8-14.8% of the genome was obtained from 80,337 BES. Annotation revealed that a large part of the genome consists of ribosomal DNA (~13.8%), followed by known transposable elements at 6.1%. Furthermore, ~7.4% of sequence was identified to harbour novel repeat elements. Homology searches against flax-ESTs and NCBI-ESTs suggested that ~5.6% of the transcriptome is unique to flax. A total of 4064 putative genomic SSRs were identified and are being developed as novel markers for their use in molecular breeding.
The first genome-wide physical map of flax constructed with BAC clones provides a framework for accessing target loci with economic importance for marker development and positional cloning. Analysis of the BES has provided insights into the uniqueness of the flax genome. Compared to other plant genomes, the proportion of rDNA was found to be very high whereas the proportion of known transposable elements was low. The SSRs identified from BES will be valuable in saturating existing linkage maps and for anchoring physical and genetic maps. The physical map and paired-end reads from BAC clones will also serve as scaffolds to build and validate the whole genome shotgun assembly.
PMCID: PMC3113786  PMID: 21554714
3.  Development and validation of a flax (Linum usitatissimum L.) gene expression oligo microarray 
BMC Genomics  2010;11:592.
Flax (Linum usitatissimum L.) has been cultivated for around 9,000 years and is therefore one of the oldest cultivated species. Today, flax is still grown for its oil (oil-flax or linseed cultivars) and its cellulose-rich fibres (fibre-flax cultivars) used for high-value linen garments and composite materials. Despite the wide industrial use of flax-derived products, and our actual understanding of the regulation of both wood fibre production and oil biosynthesis more information must be acquired in both domains. Recent advances in genomics are now providing opportunities to improve our fundamental knowledge of these complex processes. In this paper we report the development and validation of a high-density oligo microarray platform dedicated to gene expression analyses in flax.
Nine different RNA samples obtained from flax inner- and outer-stems, seeds, leaves and roots were used to generate a collection of 1,066,481 ESTs by massive parallel pyrosequencing. Sequences were assembled into 59,626 unigenes and 48,021 sequences were selected for oligo design and high-density microarray (Nimblegen 385K) fabrication with eight, non-overlapping 25-mers oligos per unigene. 18 independent experiments were used to evaluate the hybridization quality, precision, specificity and accuracy and all results confirmed the high technical quality of our microarray platform. Cross-validation of microarray data was carried out using quantitative qRT-PCR. Nine target genes were selected on the basis of microarray results and reflected the whole range of fold change (both up-regulated and down-regulated genes in different samples). A statistically significant positive correlation was obtained comparing expression levels for each target gene across all biological replicates both in qRT-PCR and microarray results. Further experiments illustrated the capacity of our arrays to detect differential gene expression in a variety of flax tissues as well as between two contrasted flax varieties.
All results suggest that our high-density flax oligo-microarray platform can be used as a very sensitive tool for analyzing gene expression in a large variety of tissues as well as in different cultivars. Moreover, this highly reliable platform can also be used for the quantification of mRNA transcriptional profiling in different flax tissues.
PMCID: PMC3091737  PMID: 20964859
4.  Gene expression analysis of flax seed development 
BMC Plant Biology  2011;11:74.
Flax, Linum usitatissimum L., is an important crop whose seed oil and stem fiber have multiple industrial applications. Flax seeds are also well-known for their nutritional attributes, viz., omega-3 fatty acids in the oil and lignans and mucilage from the seed coat. In spite of the importance of this crop, there are few molecular resources that can be utilized toward improving seed traits. Here, we describe flax embryo and seed development and generation of comprehensive genomic resources for the flax seed.
We describe a large-scale generation and analysis of expressed sequences in various tissues. Collectively, the 13 libraries we have used provide a broad representation of genes active in developing embryos (globular, heart, torpedo, cotyledon and mature stages) seed coats (globular and torpedo stages) and endosperm (pooled globular to torpedo stages) and genes expressed in flowers, etiolated seedlings, leaves, and stem tissue. A total of 261,272 expressed sequence tags (EST) (GenBank accessions LIBEST_026995 to LIBEST_027011) were generated. These EST libraries included transcription factor genes that are typically expressed at low levels, indicating that the depth is adequate for in silico expression analysis. Assembly of the ESTs resulted in 30,640 unigenes and 82% of these could be identified on the basis of homology to known and hypothetical genes from other plants. When compared with fully sequenced plant genomes, the flax unigenes resembled poplar and castor bean more than grape, sorghum, rice or Arabidopsis. Nearly one-fifth of these (5,152) had no homologs in sequences reported for any organism, suggesting that this category represents genes that are likely unique to flax. Digital analyses revealed gene expression dynamics for the biosynthesis of a number of important seed constituents during seed development.
We have developed a foundational database of expressed sequences and collection of plasmid clones that comprise even low-expressed genes such as those encoding transcription factors. This has allowed us to delineate the spatio-temporal aspects of gene expression underlying the biosynthesis of a number of important seed constituents in flax. Flax belongs to a taxonomic group of diverse plants and the large sequence database will allow for evolutionary studies as well.
PMCID: PMC3107784  PMID: 21529361
5.  Locus-specific view of flax domestication history 
Ecology and Evolution  2012;2(1):139-152.
Crop domestication has been inferred genetically from neutral markers and increasingly from specific domestication-associated loci. However, some crops are utilized for multiple purposes that may or may not be reflected in a single domestication-associated locus. One such example is cultivated flax (Linum usitatissimum L.), the earliest oil and fiber crop, for which domestication history remains poorly understood. Oil composition of cultivated flax and pale flax (L. bienne Mill.) indicates that the sad2 locus is a candidate domestication locus associated with increased unsaturated fatty acid production in cultivated flax. A phylogenetic analysis of the sad2 locus in 43 pale and 70 cultivated flax accessions established a complex domestication history for flax that has not been observed previously. The analysis supports an early, independent domestication of a primitive flax lineage, in which the loss of seed dispersal through capsular indehiscence was not established, but increased oil content was likely occurred. A subsequent flax domestication process occurred that probably involved multiple domestications and includes lineages that contain oil, fiber, and winter varieties. In agreement with previous studies, oil rather than fiber varieties occupy basal phylogenetic positions. The data support multiple paths of flax domestication for oil-associated traits before selection of the other domestication-associated traits of seed dispersal loss and fiber production. The sad2 locus is less revealing about the origin of winter tolerance. In this case, a single domestication-associated locus is informative about the history of domesticated forms with the associated trait while partially informative on forms less associated with the trait.
PMCID: PMC3297184  PMID: 22408732
Crop domestication; flax; network analysis; sad2 gene; sequence variation
6.  Efficacy of glycerol and flax seed oil as anti-adhesive barriers after thyroidectomy 
We evaluated the effects of local flax seed oil and glycerol application for reducing adhesion formation after thyroidectomy.
We randomly assigned 18 female Wistar albino rats (median weight: 275 g, median age: 4.5 mth) to 3 groups: 0.1 ml 0.9% NaCl, glycerol, and flax seed oil were sprayed in a perithyroidal area after thyroidectomy operation on all animals as anti-adhesive barriers. Rats were sacrificed on the postoperative 14th day and adhesions were evaluated macroscopically and microscopically.
The median macroscopic adhesion score was 3.0±0.0 in the 0.9% NaCl group, 1.33±0.52 in the glycerol group, and 1.67±0.53 in the flax seed oil group. The median histopathological fibrosis scores were 2.33±0.82, 0.67±0.52, and 0.83±0.75, respectively. Both glycerol and flaxseed oil group macroscopic and microscopic scores were significantly lower than the 0.9% NaCl group (p<0.05). There was no significant difference among the groups (p>0.05).
Glycerol and flax seed oil both decrease the incidence of post-thyroidectomy adhesion in rats, but glycerol is more effective.
PMCID: PMC4085115  PMID: 24973306
Glycerol - therapeutic use; Linseed Oil - therapeutic use; Thyroidectomy - adverse effects; Tissue Adhesions - complications; Tissue Adhesions - prevention & control
7.  Evaluation of Antiseizure Activity of Essential Oil from Roots of Angelica archangelica Linn. in Mice 
In the present study, the effect of essential oil of the root of Angelica archangelica Linn. was evaluated against electrically and chemically induced seizures. The seizures were induced in mice by maximal electroshock and pentylenetetrazol. The effect of essential oil of the root of Angelica archangelica on seizures was compared with standard anticonvulsant agents, phenytoin and diazepam. The essential oil of the root of Angelica archangelica suppressed duration of tonic convulsions and showed recovery in maximal electroshock induced seizures while it delayed time of onset of clonic convulsions and showed mortality protection in pentylenetetrazol induced seizures. The essential oil of the root of Angelica archangelica also produced motor impairment at the antiseizure doses. The study indicated that the essential oil exhibited antiseizure effect. The antiseizure effect may be attributed to the presence of terpenes in the essential oil.
PMCID: PMC3003174  PMID: 21188050
Angelica archangelica (Umbelliferae); convulsions; pentylenetetrazol; medicinal plants; maximal electroshock
8.  The biomedical potential of genetically modified flax seeds overexpressing the glucosyltransferase gene 
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.
PMCID: PMC3640942  PMID: 23228136
Flax; Seedcake; Phenylpropanoids; Fibroblasts
9.  Population-based resequencing revealed an ancestral winter group of cultivated flax: implication for flax domestication processes 
Ecology and Evolution  2012;2(3):622-635.
Cultivated flax (Linum usitatissimum L.) is the earliest oil and fiber crop and its early domestication history may involve multiple events of domestication for oil, fiber, capsular indehiscence, and winter hardiness. Genetic studies have demonstrated that winter cultivated flax is closely related to oil and fiber cultivated flax and shows little relatedness to its progenitor, pale flax (L. bienne Mill.), but winter hardiness is one major characteristic of pale flax. Here, we assessed the genetic relationships of 48 Linum samples representing pale flax and four trait-specific groups of cultivated flax (dehiscent, fiber, oil, and winter) through population-based resequencing at 24 genomic regions, and revealed a winter group of cultivated flax that displayed close relatedness to the pale flax samples. Overall, the cultivated flax showed a 27% reduction of nucleotide diversity when compared with the pale flax. Recombination frequently occurred at these sampled genomic regions, but the signal of selection and bottleneck was relatively weak. These findings provide some insight into the impact and processes of flax domestication and are significant for expanding our knowledge about early flax domestication, particularly for winter hardiness.
PMCID: PMC3399149  PMID: 22822439
Crop domestication; cultivated flax; pale flax; sequence variation; winter hardiness
10.  Anticonvulsant potential of ethanol extracts and their solvent partitioned fractions from Flemingia strobilifera root 
Pharmacognosy Research  2013;5(4):265-270.
Flemingia strobilifera (FS) R.Br. (Fabaceae) is an important medicinal plant. In wealth of India it has been reported that roots of FS are used by santals in epilepsy, hysteria, insomnia, and to relieve pain. In Burma also the roots of F. strobilifera are used to treat epilepsy.
To investigate anticonvulsant potential of 95% ethanol extract and four subsequent fractions (petroleum ether, chloroform, ethyl acetate, and aqueous fractions of the roots of FS against pentylenetetrazole (PTZ) and maximal electroshock (MES) induced convulsions.
Material and Methods:
All the fractions and crude ethanol extract were administered (i.e., 200, 400, 600 mg/kg, p.o.) for 7 days and at the end of the treatment convulsions were induced experimentally using pentylenetetrazole and Maximal electroshock Test. Diazepam and phenytoin (4 mg/kg, i.p. and 20 mg/kg, i.p., respectively) were used as reference anticonvulsant drugs against experimentally induced convulsions. The latency of tonic convulsions and the numbers of animals protected from tonic convulsions were noted.
High doses (200 and 300 mg/kg, p.o.) of ethyl acetate fraction and 95% ethanol crude extract (400 and 600 mg/kg, p.o.) significantly reduced the duration of seizure induced by maximal electroshock (MES). The same dose also protected from pentylenetetrzole-induced tonic seizures and significantly delayed the onset of tonic seizures. However, pet, ether, chloroform, and aqueous fraction at any of the doses used (i.e., 100, 200, 300 mg/kg, p.o.) did not show any significant effect on PTZ and MES induced convulsions. The treatment with crude ethanolic extract and ethyl acetate fraction caused signs of central nervous system depressant action in the locomotor activity test, confirmed by the potentiation of sodium pentobarbital sleeping time. Both did not cause disturbance in motor coordination assessed by rotarod test.
The data suggest that crude ethanol extract and ethyl acetate fraction of roots of Flemingia strobilifera have a central nervous system depressant action and behave as a potential anticonvulsant. It may produce its anticonvulsant effect via non-specific mechanism since it reduced the duration of seizures produced by maximal electroshock as well as delayed the latency of seizures produced by pentylenetetrazole.
PMCID: PMC3807991  PMID: 24174820
Antiepileptic; Flemingia strobilifera;  maximal electroshock; pentylenetetrazole; seizures
11.  Anticonvulsant Effect of Cicer arietinum Seed in Animal Models of Epilepsy: Introduction of an Active Molecule with Novel Chemical Structure 
Iranian Biomedical Journal  2015;19(1):45-50.
Background: Cicer arietinum (Chickpea) is one of the most important harvests in the world with high nutritional value. Lack of essential oils in the seeds of Chickpea is an advantage in search for drug-like molecules with less toxicity. We evaluated anticonvulsant effect of C. arietinum in common animal models of epilepsy. Methods: Dichloromethane extract was obtained from C. arietinum seeds by percolation. Acute toxicity of the extract was assessed in mice. Protective effect of the extract was examined against tonic seizures induced by maximal electroshock (MES; 50 mA, 50 Hz, 1 s) in mice, clonic seizures induced by pentylenetetrazole (PTZ; 60 mg/kg, i.p.) in mice, and electrical kindling model of complex partial seizures in rats. The extract was fractionated by n-hexane to f1 and f2 fractions. The extract and fractions underwent phytochemical analysis by thin layer chromatography. The active anticonvulsant fraction, f1, was subjected to matrix assisted laser desorption/ionization (MALDI) mass analysis. Results: The crude extract had neither toxicity up to 7 g/kg nor protective activity in MES and kindling models. However, it significantly inhibited clonic seizures induced by PTZ. f1 fraction mimicked protective effect of the extract. Phytochemical screening revealed the presence of considerable amount of alkaloids in the extract and fractions. Moreover, a novel structural class was detected in f1 fraction. Conclusion: Finding an anticonvulsant molecule pertaining to a new structural class in the seeds of C. arietinum promises an effective and inexpensive source of antiepileptic medication. Further studies are needed to identify its mechanism of action and more clues into its structure-activity relationship.
PMCID: PMC4322232  PMID: 25605489
Anticonvulsants; Cicer; Kindling; Pentylenetetrazole (PTZ)
12.  PT-Flax (phenotyping and TILLinG of flax): development of a flax (Linum usitatissimum L.) mutant population and TILLinG platform for forward and reverse genetics 
BMC Plant Biology  2013;13:159.
Flax (Linum usitatissimum L.) is an economically important fiber and oil crop that has been grown for thousands of years. The genome has been recently sequenced and transcriptomics are providing information on candidate genes potentially related to agronomically-important traits. In order to accelerate functional characterization of these genes we have generated a flax EMS mutant population that can be used as a TILLinG (Targeting Induced Local Lesions in Genomes) platform for forward and reverse genetics.
A population of 4,894 M2 mutant seed families was generated using 3 different EMS concentrations (0.3%, 0.6% and 0.75%) and used to produce M2 plants for subsequent phenotyping and DNA extraction. 10,839 viable M2 plants (4,033 families) were obtained and 1,552 families (38.5%) showed a visual developmental phenotype (stem size and diameter, plant architecture, flower-related). The majority of these families showed more than one phenotype. Mutant phenotype data are organised in a database and can be accessed and searched at UTILLdb ( Preliminary screens were also performed for atypical fiber and seed phenotypes. Genomic DNA was extracted from 3,515 M2 families and eight-fold pooled for subsequent mutant detection by ENDO1 nuclease mis-match cleavage. In order to validate the collection for reverse genetics, DNA pools were screened for two genes coding enzymes of the lignin biosynthesis pathway: Coumarate-3-Hydroxylase (C3H) and Cinnamyl Alcohol Dehydrogenase (CAD). We identified 79 and 76 mutations in the C3H and CAD genes, respectively. The average mutation rate was calculated as 1/41 Kb giving rise to approximately 9,000 mutations per genome. Thirty-five out of the 52 flax cad mutant families containing missense or codon stop mutations showed the typical orange-brown xylem phenotype observed in CAD down-regulated/mutant plants in other species.
We have developed a flax mutant population that can be used as an efficient forward and reverse genetics tool. The collection has an extremely high mutation rate that enables the detection of large numbers of independant mutant families by screening a comparatively low number of M2 families. The population will prove to be a valuable resource for both fundamental research and the identification of agronomically-important genes for crop improvement in flax.
PMCID: PMC3853753  PMID: 24128060
Flax; TILLinG; Mutants; Fiber; Lignin; Lignan; Oil; Fatty acids
13.  Identification, characterization and distribution of transposable elements in the flax (Linum usitatissimum L.) genome 
BMC Genomics  2012;13:644.
Flax (Linum usitatissimum L.) is an important crop for the production of bioproducts derived from its seed and stem fiber. Transposable elements (TEs) are widespread in plant genomes and are a key component of their evolution. The availability of a genome assembly of flax (Linum usitatissimum) affords new opportunities to explore the diversity of TEs and their relationship to genes and gene expression.
Four de novo repeat identification algorithms (PILER, RepeatScout, LTR_finder and LTR_STRUC) were applied to the flax genome assembly. The resulting library of flax repeats was combined with the RepBase Viridiplantae division and used with RepeatMasker to identify TEs coverage in the genome. LTR retrotransposons were the most abundant TEs (17.2% genome coverage), followed by Long Interspersed Nuclear Element (LINE) retrotransposons (2.10%) and Mutator DNA transposons (1.99%). Comparison of putative flax TEs to flax transcript databases indicated that TEs are not highly expressed in flax. However, the presence of recent insertions, defined by 100% intra-element LTR similarity, provided evidence for recent TE activity. Spatial analysis showed TE-rich regions, gene-rich regions as well as regions with similar genes and TE density. Monte Carlo simulations for the 71 largest scaffolds (≥ 1 Mb each) did not show any regional differences in the frequency of TE overlap with gene coding sequences. However, differences between TE superfamilies were found in their proximity to genes. Genes within TE-rich regions also appeared to have lower transcript expression, based on EST abundance. When LTR elements were compared, Copia showed more diversity, recent insertions and conserved domains than the Gypsy, demonstrating their importance in genome evolution.
The calculated 23.06% TE coverage of the flax WGS assembly is at the low end of the range of TE coverages reported in other eudicots, although this estimate does not include TEs likely found in unassembled repetitive regions of the genome. Since enrichment for TEs in genomic regions was associated with reduced expression of neighbouring genes, and many members of the Copia LTR superfamily are inserted close to coding regions, we suggest Copia elements have a greater influence on recent flax genome evolution while Gypsy elements have become residual and highly mutated.
PMCID: PMC3544724  PMID: 23171245
Transposable elements; Flax; Genome evolution; LTR elements; Gene expression
14.  Effects of Hydro-alcoholic Extract of Anethum Graveolens Seed on Pentylenetetrazol-induced Seizure in Adult Male Mice 
Basic and Clinical Neuroscience  2014;5(3):199-204.
Regarding chronic nature of epilepsy and its side effects and to access the effective treatment procedures, herbal medicine has received remarkable interest. The aim of this study was to determine the anticonvulsant effects of hydro-alcoholic extract of Anethum graveolens seed on pentylenetetrazol (PTZ) -induced seizure in male mice.
Fifty-six albino male mice were divided randomly into seven groups including the negative control (saline), positive control (Phenobarbital) and treatment groups using different doses of hydro-alcoholic extract of Anethum graveolens seed (50, 100, 300, 500 and 1000 mg/ kg). To provoke convulsion, PTZ was injected to all groups and initiation time of myoclonic and tonic-clonic seizures as well as surveillance after 24 h were measured.
The results indicated that hydro-alcoholic extract of Anethum graveolens seed (AGS) delayed the initiation time of myoclonic and tonic-clonic seizures in comparison with saline group. The latency was considerable for myoclonic and tonic-clonic seizures at all above mentioned doses of AGS extract except for the lowest one. Moreover, the protective effect of AGS extract against mortality was statistically significant at all doses except for 50 mg/kg.
As the hydro-alcoholic extract of AGS showed an appropriate response in experimental model of convulsion, it might be considered as an adjuvant therapy with other traditional antiepileptic medications.
PMCID: PMC4202545  PMID: 25337380
Anethum Graveolens; Pentylenetetrazol-induced Seizure; Mice
15.  PA01.73. A retrospective analysis of efficacy of various ayurvedic formulations in psychiatric diseases 
Ancient Science of Life  2012;32(Suppl 1):S123.
Mental disorders are high on rise in India. Epidemiological studies conducted in India on mental and behavioural disorders report varying prevalence rates, ranging from 9.5 to 370 per 1000 population. The overall DALYs burden for mental disorders is projected to increase to 15 per cent by the year 2020 and this increase is proportionately larger than that for cardiovascular diseases. Ayurveda bears great responsibility in preventing and treating the mental disorders. Medhya Rasayana is the treatment perspective to prevent and manage psychiatric disorders. In Ayurveda, Rasayana therapy has been stated as a unique therapy in curing mental diseases. It can promote memory and intelligence and can increase immunity against disease and promote strength and vitality as well as it can control ageing process by serving as anti oxidant agent. A review of various studies carried out in Ayurveda is made to enlist the best effective treatment measures in promoting and preventing mental disorders.
Available research works carried out at Institute for Post Graduate Teaching & Research in Ayurveda, Jamnagar from the year 2001 to 2012 are screened and referred with relation to mental disorders. The treatments are reviewed and enlisted for assessing the efficacy.
On reviewing the researches, it was found that nearly 15 works found suitable are carried out in relation to mental disorders. The data shows that Ayurvedic formulations like Aamalakyadi and Medhya Rasayana Ghrita (in Alzheimer's disease), Rasayana Ghana tablets (in stress), Rasona tablets, Brahmi ghrita, flax seed capsules and Ashwagandharishta (in depression), Shirodhara (in insomnia), Saraswatarishta (in perimenopausal syndrome) are effective in psychiatric diseases.
Researches show that Ayurvedic formulations are effective in moderate manner in treating the psychiatric diseases. Ayurvedic Medhya Rasayana formulations can be used for preventing and managing psychiatric disorders.
PMCID: PMC3800878
16.  Role of cinnarizine and nifedipine on anticonvulsant effect of sodium valproate and carbamazepine in maximal electroshock and pentylenetetrazole model of seizures in mice 
To study the effect of calcium channel blockers (CCBs) cinnarizine and nifedipine on maximal electroshock (MES)-induced and pentylenetetrazole (PTZ)-induced convulsions and also their effect in combination with conventional antiepileptic drugs (CAED).
Materials and Methods:
For this study, Swiss albino mice were used. Effects of cinnarizine (30 mg/kg), nifedipine (5 mg/kg), sodium valproate (300 mg/kg) and carbamazepine (8 mg/kg) alone and in combination were studied in MES and PTZ seizure models. Abolition of hind limb tonic extension was an index of anticonvulsant activity in MES, while for PTZ seizures, failure to observe even a single episode of tonic spasm for 5 s duration for 1 h was the index. With this, percentage protection was calculated and statistical analysis was carried out using Fisher’s exact test (Ovvind Langsrud software, German version).
In MES seizures, augmented effects were obtained when cinnarizine was combined with sodium valproate, i.e. 100%. In PTZ-induced seizures, augmented effects were obtained when nifedipine was combined with sodium valproate, i.e. 100%. Thus, cinnarizine added to sodium valproate therapy produces significant protection against MES seizures while nifedipine added to sodium valproate therapy produces significant protection against PTZ seizures.
The results provide a lead for potential benefit of adding CCBs to sodium valproate in the treatment of epilepsy, which needs to be explored further.
PMCID: PMC3043329  PMID: 21350614
Carbamazepine; cinnarizine; nifedipine; seizures; sodium valproate
17.  Plausible antioxidant biomechanics and anticonvulsant pharmacological activity of brain-targeted β-carotene nanoparticles 
β-Carotene has been established as a known free radical scavenger with chain-breaking antioxidant properties. It has been documented for the treatment of epileptic convulsions at a 200 mg/kg body weight dose. The reported pathogenesis for epileptic convulsions is oxidative stress. Hence, experimental epileptic convulsions via oxidative stress was induced in albino mice epileptic models (maximal electroshock seizure and pentylenetetrazole [PTZ]). A dose concentration equivalent to 2 mg/kg was efficaciously administered in the form of brain-targeted polysorbate-80-coated poly(d,l-lactide-co-glycolide) nanoparticles. The nanoparticles were prepared by solvent evaporation technique and further characterized for their physical parameters, in-vitro release kinetics, and in-vivo brain release via various standard methods. Normal β-carotene nanoparticles (BCNP) and polysorbate-80-coated β-carotene nanoparticles (P-80-BCNP) of 169.8 ± 4.8 nm and 176.3 ± 3.2 nm in size, respectively, were formulated and characterized. Their zeta potential and polydispersity index were subsequently evaluated after 5 months of storage to confirm stability. In vivo activity results showed that a 2 mg unformulated β-carotene dose was ineffective as an anticonvulsant. However, salutary response was reported from BCNP at the same dose, as the hind limb duration decreased significantly in maximal electroshock seizure to 9.30 ± 0.86 seconds, which further decreased with polysorbate-80 coating to 2.10 ± 1.16 seconds as compared to normal control (15.8 ± 1.49 seconds) and placebo control (16.50 ± 1.43 seconds). In the PTZ model, the duration of general tonic–clonic seizures reduced significantly to 2.90 ± 0.98 seconds by the use of BCNP and was further reduced on P-80-BCNP to 1.20 ± 0.20 seconds as compared to PTZ control and PTZ-placebo control (8.09 ± 0.26 seconds). General tonic–clonic seizures latency was increased significantly to 191.0 ± 9.80 seconds in BCNP and was further increased in P-80-BCNP to 231.0 ± 16.30 seconds, as compared to PTZ (120.10 ± 4.50 seconds) and placebo control (120.30 ± 7.4 seconds). The results of this study demonstrate a plausible novel anticonvulsant activity of β-carotene at a low dose of 2 mg/kg, with brain-targeted nanodelivery, thus increasing its bioavailability and stability.
PMCID: PMC3419510  PMID: 22915852
anticonvulsant; blood–brain barrier (BBB); targeted brain delivery; polysorbate-80-coated β-carotene nanoparticles (P-80-BCNP); maximal electroshock seizure (MES); pentylenetetrazole (PTZ)
18.  Phylogenomic analysis of UDP glycosyltransferase 1 multigene family in Linum usitatissimum identified genes with varied expression patterns 
BMC Genomics  2012;13:175.
The glycosylation process, catalyzed by ubiquitous glycosyltransferase (GT) family enzymes, is a prevalent modification of plant secondary metabolites that regulates various functions such as hormone homeostasis, detoxification of xenobiotics and biosynthesis and storage of secondary metabolites. Flax (Linum usitatissimum L.) is a commercially grown oilseed crop, important because of its essential fatty acids and health promoting lignans. Identification and characterization of UDP glycosyltransferase (UGT) genes from flax could provide valuable basic information about this important gene family and help to explain the seed specific glycosylated metabolite accumulation and other processes in plants. Plant genome sequencing projects are useful to discover complexity within this gene family and also pave way for the development of functional genomics approaches.
Taking advantage of the newly assembled draft genome sequence of flax, we identified 137 UDP glycosyltransferase (UGT) genes from flax using a conserved signature motif. Phylogenetic analysis of these protein sequences clustered them into 14 major groups (A-N). Expression patterns of these genes were investigated using publicly available expressed sequence tag (EST), microarray data and reverse transcription quantitative real time PCR (RT-qPCR). Seventy-three per cent of these genes (100 out of 137) showed expression evidence in 15 tissues examined and indicated varied expression profiles. The RT-qPCR results of 10 selected genes were also coherent with the digital expression analysis. Interestingly, five duplicated UGT genes were identified, which showed differential expression in various tissues. Of the seven intron loss/gain positions detected, two intron positions were conserved among most of the UGTs, although a clear relationship about the evolution of these genes could not be established. Comparison of the flax UGTs with orthologs from four other sequenced dicot genomes indicated that seven UGTs were flax diverged.
Flax has a large number of UGT genes including few flax diverged ones. Phylogenetic analysis and expression profiles of these genes identified tissue and condition specific repertoire of UGT genes from this crop. This study would facilitate precise selection of candidate genes and their further characterization of substrate specificities and in planta functions.
PMCID: PMC3412749  PMID: 22568875
19.  A New Derivative of Valproic Acid Amide Possesses a Broad-spectrum Antiseizure Profile and Unique Activity Against Status Epilepticus and Organophosphate Neuronal Damage 
Epilepsia  2011;53(1):134-146.
sec-Butyl-propylacetamide (SPD) is a one-carbon homologue of valnoctamide (VCD), a CNS-active amide derivative of valproic acid (VPA) currently in phase II clinical trials. The current study evaluated the anticonvulsant activity of SPD in a battery of rodent seizure and epilepsy models and assessed its efficacy in rat and guinea pig models of status epilepticus (SE) and neuroprotection in an organotypic hippocampal slice model of excitotoxic cell death.
SPD’s anticonvulsant activity was evaluated in several rodent seizure and epilepsy models including: maximal electroshock (MES), 6Hz psychomotor, subcutaneous (s.c.) metrazol-, s.c., picrotoxin, s.c. bicuculline, audiogenic and corneal and hippocampal kindled seizures following intraperitoneal administration. Results obtained with SPD are discussed in relationship to those obtained with VPA and VCD. SPD was also evaluated for its ability to block benzodiazepine-resistant SE induced by pilocarpine (rats) and soman (rats and guinea pigs) following intraperitoneal administration. SPD was tested for its ability to block excitotoxic cell death induced by the glutamate agonists N-methyl-D-Aspartate (NMDA) and kainic acid (KA) using organotypic hippocampal slices and SE-induced hippocampal cell death using FluoroJade B staining. The cognitive function of SPD-treated rats that were protected against pilocarpine-induced convulsive SE was examined 10-14 days post SE using the Morris water maze (MWM). The relationship between the pharmacokinetic profile of SPD and its efficacy against soman-induced SE was evaluated in two parallel studies following SPD (60 mg/kg, i.p.) administration in the soman SE rat model.
Key Findings
SPD was highly effective and displayed a wide protective index (PI=TD50/ED50) in the standardized seizure and epilepsy models employed. SPD’s wide PI values demonstrate that it is effective at doses well below those that produce behavioral impairment. Unlike VCD, SPD also displayed anticonvulsant activity in the rat pilocarpine model of SE. Thirty minutes after the induction of SE, the calculated rat-ED50 for SPD against convulsive SE in this model was 84mg/kg. SPD was not neuroprotective in the organotypic hippocampal slice preparation; however, it did display hippocampal neuroprotection in both SE models and cognitive sparing in the MWM which was associated with its antiseizure effect against pilocarpine-induced SE. When administered 20 and 40min after SE onset, SPD (100-174mg/kg) produced long-lasting efficacy (e.g., 4-8hr) against soman-induced convulsive and electrographic SE in both rats and guinea pigs. SPD-ED50 values in guinea pigs were 67mg/kg and 92mg/kg at when administered at SE onset or 40min after SE onset, respectively. Assuming linear PK, the PK-PD results (rats) suggests that effective SPD plasma levels ranged between 8-40mg/L (20 min post onset of soman-induced seizures) and 12-50mg/L (40 min post onset of soman-induced seizures). The time to peak (tmax) pharmacodynamic effect (PD-tmax) occurred after the PK-tmax thereby suggesting that SPD undergoes slow distribution to extra-plasmatic sites likely responsible for SPD’s antiseizure activity.
The results demonstrate that SPD is a broad-spectrum antiseizure compound that blocks SE induced by pilocarpine and soman and affords in vivo neuroprotection that is associated with cognitive sparing. Its activity against SE is superior to diazepam in terms of rapid onset, potency and its effect on animal mortality and functional improvement.
PMCID: PMC3253224  PMID: 22150444
New anticonvulsant drug; Status epilepticus (SE); pilocarpine-induced SE rat model; Soman-induced SE rat model; Pharmacokinetic-pharmacodynamic correlation
20.  Pollen-mediated gene flow in flax (Linum usitatissimum L.): can genetically engineered and organic flax coexist? 
Heredity  2010;106(4):557-566.
Coexistence allows growers and consumers the choice of producing or purchasing conventional or organic crops with known standards for adventitious presence of genetically engineered (GE) seed. Flax (Linum usitatissimum L.) is multipurpose oilseed crop in which product diversity and utility could be enhanced for industrial, nutraceutical and pharmaceutical markets through genetic engineering. If GE flax were released commercially, pollen-mediated gene flow will determine in part whether GE flax could coexist without compromising other markets. As a part of pre-commercialization risk assessment, we quantified pollen-mediated gene flow between two cultivars of flax. Field experiments were conducted at four locations during 2006 and 2007 in western Canada using a concentric donor (20 × 20 m) receptor (120 × 120 m) design. Gene flow was detected through the xenia effect of dominant alleles of high α-linolenic acid (ALA; 18:3cisΔ9,12,15) to the low ALA trait. Seeds were harvested from the pollen recipient plots up to a distance of 50 m in eight directions from the pollen donor. High ALA seeds were identified using a thiobarbituric acid test and served as a marker for gene flow. Binomial distribution and power analysis were used to predict the minimum number of seeds statistically required to detect the frequency of gene flow at specific α (confidence interval) and power (1−β) values. As a result of the low frequency of gene flow, approximately 4 million seeds were screened to derive accurate quantification. Frequency of gene flow was highest near the source: averaging 0.0185 at 0.1 m but declined rapidly with distance, 0.0013 and 0.00003 at 3 and 35 m, respectively. Gene flow was reduced to 50% (O50) and 90% (O90) between 0.85 to 2.64 m, and 5.68 to 17.56 m, respectively. No gene flow was detected at any site or year >35 m distance from the pollen source, suggesting that frequency of gene flow was ⩽0.00003 (P=0.95). Although it is not possible to eliminate all adventitious presence caused by pollen-mediated gene flow, through harvest blending and the use of buffer zones between GE and conventional flax fields, it could be minimized. Managing other sources of adventitious presence including seed mixing and volunteer populations may be more problematic.
PMCID: PMC3183899  PMID: 20551976
coexistence; adventitious presence; conventional; organic; pollen-mediated gene flow; regulation
21.  Effects of chronic administration of gabapentin and carbamazepine on the histomorphology of the hippocampus and striatum 
Annals of Neurosciences  2014;21(2):57-61.
Antiepileptic drugs used to avert epileptic seizures necessitate prolonged duration for improved efficacy and could induce some side effects.
The present study investigated the effect of chronic administration of two common antiepileptic drugs – gabapentin and carbamazepine, on the histomorphology of the hippocampus and striatum in adult rats.
25 adult male Wistar rats were grouped randomly into 5 groups. 3 groups were administered either therapeutic doses of gabapentin (16 mg/kg) or carbamazepine (20 mg/kg) or sub-therapeutic dose of gabapentin plus carbamazepine (8 + 10 mg/kg). To confirm anticonvulsant effects, these animals were kindled for seizures at sub-maximal electroshock. Appropriate negative and positive controls were given normal saline. At the end of treatment, brain tissues were obtained and processed for histological procedures.
The study confirm significant decrease (P<0.001) in convulsion parameters tonic flexion, tonic extension and clonic convulsion, between drug treated groups and electroshock control. Histological studies revealed significant increase (p<0.001) in neurons showing features of degeneration in the hippocampus, for drug treated groups as compared to normal and electroshock control. Also, drug treatment reduced nissl activity in both hippocampus and striatum.
Chronic administration of gabapentin and carbamazepine may cause increase in neurodegenerative changes in the adult brain.
PMCID: PMC4117164  PMID: 25206062
Epilepsy; Antiepileptic drugs; Histology; Brain; Rats
22.  Anticonvulsant Effect of Antiaris toxicaria (Pers.) Lesch. (Moraceae) Aqueous Extract in Rodents 
ISRN Pharmacology  2013;2013:519208.
Antiaris toxicaria (Moraceae) was evaluated for anticonvulsant activity in rodents. Animal models used include maximal electroshock test (MEST); pentylenetetrazole-induced (PTZ) convulsions; picrotoxin-induced (PCT) convulsions; strychnine- (STR-) and 4-aminopyridine-induced convulsions. Increase in latency to seizures as well as reduction in duration and frequency of seizures indicated anticonvulsant activity. The extract was more effective in all models used except the maximal electroshock test and strychnine-induced convulsions. Antiaris toxicaria aqueous extract (200, 400, and 800 mg kg−1) significantly (P < 0.05 − 0.01) shortened the duration of convulsions in PTZ- and PCT-induced seizures. Delay in the onset of convulsions in the two tests was significant (P < 0.001). Reduction in the frequency of seizures was also significant (P < 0.05 − 0.001) in both tests. Antiaris further delayed the onset of seizures in 4-aminopyridine model while producing 75% protection against death in mice. Diazepam (0.1, 0.3, and 1 mg kg−1), carbamazepine (3, 10, and 30 mg kg−1), and sodium valproate (100–400 mg kg−1) were used as reference anticonvulsant drugs for various models. Flumazenil blocked the effect of the extract in the PTZ test significantly suggesting that Antiaris toxicaria may be acting by enhancing the effects of the GABAergic system. Antiaris toxicaria aqueous extract therefore possesses anticonvulsant activity.
PMCID: PMC3791639  PMID: 24167736
23.  Selection of reference genes for quantitative gene expression normalization in flax (Linum usitatissimum L.) 
BMC Plant Biology  2010;10:71.
Quantitative real-time PCR (qRT-PCR) is currently the most accurate method for detecting differential gene expression. Such an approach depends on the identification of uniformly expressed 'housekeeping genes' (HKGs). Extensive transcriptomic data mining and experimental validation in different model plants have shown that the reliability of these endogenous controls can be influenced by the plant species, growth conditions and organs/tissues examined. It is therefore important to identify the best reference genes to use in each biological system before using qRT-PCR to investigate differential gene expression. In this paper we evaluate different candidate HKGs for developmental transcriptomic studies in the economically-important flax fiber- and oil-crop (Linum usitatissimum L).
Specific primers were designed in order to quantify the expression levels of 20 different potential housekeeping genes in flax roots, internal- and external-stem tissues, leaves and flowers at different developmental stages. After calculations of PCR efficiencies, 13 HKGs were retained and their expression stabilities evaluated by the computer algorithms geNorm and NormFinder. According to geNorm, 2 Transcriptional Elongation Factors (TEFs) and 1 Ubiquitin gene are necessary for normalizing gene expression when all studied samples are considered. However, only 2 TEFs are required for normalizing expression in stem tissues. In contrast, NormFinder identified glyceraldehyde-3-phosphate dehydrogenase (GADPH) as the most stably expressed gene when all samples were grouped together, as well as when samples were classed into different sub-groups.
qRT-PCR was then used to investigate the relative expression levels of two splice variants of the flax LuMYB1 gene (homologue of AtMYB59). LuMYB1-1 and LuMYB1-2 were highly expressed in the internal stem tissues as compared to outer stem tissues and other samples. This result was confirmed with both geNorm-designated- and NormFinder-designated-reference genes.
The use of 2 different statistical algorithms results in the identification of different combinations of flax HKGs for expression data normalization. Despite such differences, the use of geNorm-designated- and NormFinder-designated-reference genes enabled us to accurately compare the expression levels of a flax MYB gene in different organs and tissues. Our identification and validation of suitable flax HKGs will facilitate future developmental transcriptomic studies in this economically-important plant.
PMCID: PMC3095345  PMID: 20403198
24.  Genome wide SNP discovery in flax through next generation sequencing of reduced representation libraries 
BMC Genomics  2012;13:684.
Flax (Linum usitatissimum L.) is a significant fibre and oilseed crop. Current flax molecular markers, including isozymes, RAPDs, AFLPs and SSRs are of limited use in the construction of high density linkage maps and for association mapping applications due to factors such as low reproducibility, intense labour requirements and/or limited numbers. We report here on the use of a reduced representation library strategy combined with next generation Illumina sequencing for rapid and large scale discovery of SNPs in eight flax genotypes. SNP discovery was performed through in silico analysis of the sequencing data against the whole genome shotgun sequence assembly of flax genotype CDC Bethune. Genotyping-by-sequencing of an F6-derived recombinant inbred line population provided validation of the SNPs.
Reduced representation libraries of eight flax genotypes were sequenced on the Illumina sequencing platform resulting in sequence coverage ranging from 4.33 to 15.64X (genome equivalents). Depending on the relatedness of the genotypes and the number and length of the reads, between 78% and 93% of the reads mapped onto the CDC Bethune whole genome shotgun sequence assembly. A total of 55,465 SNPs were discovered with the largest number of SNPs belonging to the genotypes with the highest mapping coverage percentage. Approximately 84% of the SNPs discovered were identified in a single genotype, 13% were shared between any two genotypes and the remaining 3% in three or more. Nearly a quarter of the SNPs were found in genic regions. A total of 4,706 out of 4,863 SNPs discovered in Macbeth were validated using genotyping-by-sequencing of 96 F6 individuals from a recombinant inbred line population derived from a cross between CDC Bethune and Macbeth, corresponding to a validation rate of 96.8%.
Next generation sequencing of reduced representation libraries was successfully implemented for genome-wide SNP discovery from flax. The genotyping-by-sequencing approach proved to be efficient for validation. The SNP resources generated in this work will assist in generating high density maps of flax and facilitate QTL discovery, marker-assisted selection, phylogenetic analyses, association mapping and anchoring of the whole genome shotgun sequence.
PMCID: PMC3557168  PMID: 23216845
Single nucleotide polymorphism (SNP); Genotyping-by-sequencing (GBS); Reduced representation library (RRL); Illumina; Flax; Linum usitatissimum; AGSNP
25.  Anticonvulsant activity of raw and classically processed Vacha (Acorus calamus Linn.) rhizomes 
Ayu  2012;33(1):119-122.
The rhizome of Vacha (Acorus calamus) has been used in Ayurvedic medicine for the treatment of various ailments, such as epilepsy, headache, eye disorders, insomnia, loss of memory, etc. Previous studies demonstrated that Vacha rhizome is having significant anticonvulsant activity against various induced seizures models in experimental animals. Ayurvedic pharmacopoeia of India has advocated Shodhana (purificatory procedures) to be done prior to its use. In the present study a comparative anticonvulsant activity of raw and Shodhita (classically processed) Vacha rhizomes were screened against Maximal Electro Shock (MES) seizure model to assess the effect of classical purificatory procedure on pharmacological action of Vacha. Phenytoin was used as standard antiepileptic drug for comparison. Pretreatment with both raw and classically processed Vacha samples exhibited significant anticonvulsant activity by decreasing the duration of tonic extensor phase. Further classically processed Vacha statistically decreased the duration of convulsion and stupor phases of MES-induced seizures. The results obtained from the present study clearly confirmed the anticonvulsant activity of raw Vacha and subjecting to classical Shodhana procedure did not alter the efficacy of Vacha rhizomes instead it enhanced the activity profile of the Vacha.
PMCID: PMC3456849  PMID: 23049196
Acorus calamus; anticonvulsant; epilepsy; phenytoin; shodhana; vacha

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