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1.  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
2.  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
3.  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
4.  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
5.  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
6.  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
7.  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
8.  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
9.  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
10.  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
11.  Aqueous Stem Bark Extract of Stereospermum Kunthianum (Cham, Sandrine Petit) Protects Against Generalized Seizures in Pentylenetetrazole and Electro-Convulsive Models in Rodents 
Stereospermum kunthianum, Cham Sandrine Petit (Bignoniaceae) known in English as pink jacaranda is used in traditional medicine to treat an array of ailments including febrile convulsions in infants and young children by the rural dwellers in Nigeria. This study examined the anticonvulsant activity of its aqueous stem bark extract (100 – 400mg/kg) against maximal electroshock and pentylenetetrazole-induced seizures in rodents. Phenobarbitone and ethosuximide were used as reference anticonvulsant drugs for comparison. Stereospermum kunthianum extract (200 – 400mg/kg, i.p.) remarkably protected (76.9% and 84.6 % respectively) the rats against electroshock-induced seizures. However, the extract (200- 400mg/kg) when administered orally showed a comparatively less effect (33.3% and 55.6% respectively) to the intraperitoneally administered extract in the maximal electroshock test. The extract (100–400mg/kg, i.p.) significantly delayed (p<0.05) the onset of pentylenetetrazole-induced clonic seizures but only slightly prolonged the time of death of the mice. Although the findings in the present study do not provide conclusive evidence, it appears that the aqueous stem bark extract of Stereospermum kunthianum produces its antiseizure effect by enhancing GABAergic neurotransmission and/or action in the brain. The results indicate that the aqueous extract possesses anticonvulsant activity in rodents and therefore tend to suggest that the shrub may be used as a natural supplementary remedy in the management, control and/or treatment of childhood convulsions. It can be concluded that the aqueous stem bark extract possesses anticonvulsant activity and therefore lend pharmacological credence to the traditionally claimed use in the treatment of childhood convulsions.
PMCID: PMC2816468  PMID: 20606775
Stereospermum kunthianum; Anticonvulsant activity; maximal Electroshock; pentylenetetra-zole
12.  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
13.  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
14.  Effect of Purificatory Measures Through Cow's Urine and Milk on Strychnine and Brucine Content of Kupeelu (Strychnos Nuxvomica Linn.) Seeds 
Strychnos nux vomica Linn.(Loganaceae) commonly known as Nux vomica (Kupeelu), is a poisonous plant and its seeds are used widely in Ayurvedic system of medicine since time immemorial. Ayurveda advocates that nux vomica seeds are to be administered in therapeutics only after going through certain purificatory measures (Shodhana). There are more than six media: cow's urine (Go mutra), cow's milk (Go dugdha), cow's ghee (Go ghrita), Kanji (thin gruel), castor oil (Eranda taila) and fresh ginger juice (Ardraka swarasa) etc., which have been reported in different classical texts of Ayurveda for proper processing of nux vomica seeds. In this study, an attempt has been made to purify the seeds by using three different methods as described in ancient treatise by using cow's urine and cow's milk as media alone and together. This study revealed that all the methods studied reduced the toxicity of strychnine and brucine contents in comparison to the raw seeds as determined by HPTLC. Out of these three methods maximum reduction in strychnine and brucine contents was found when the seeds were purified by keeping them in cow's urine for seven days followed by boiling in cow's milk for three hrs.
PMCID: PMC3746529  PMID: 23983327
Kupeelu; Strychnos nuxvomica; Shodhana; strychnine; Ayurveda; brucine; Cow's milk; Cow's urine
15.  Effect of Ethanolic Extract of Indigofera tinctoria on Chemically-Induced Seizures and Brain GABA Levels in Albino Rats 
Indigofera tinctoria Linn. of Fabaceae family is claimed to be useful to control epileptic seizures in the Indian system of folkore medicine. This study was designed to evaluate tinctoria and to verify the claim.
Materials and Methods
Seizures were induced in male albino rats with pentylenetetrazole (PTZ). The test group animals were administered ethanolic extract of Indigofera tinctoria (EEIT) orally. The time of onset and duration of clonic convulsions were recorded. Maximal electroshock seizures (MES) were induced in animals. The duration of hind limb tonic extension (HLTE) was recorded. GABA levels and GABA transaminase activity in brain were estimated.
In PTZ model, EEIT significantly (P< 0.01, P< 0.001) delayed the onset of convulsions and reduced the duration of seizures in a dose dependent manner. A significant (P< 0.05) reduction in the duration of HLTE at higher doses of EEIT was observed in MES model. Increase in brain GABA levels was observed on treatment with EEIT at 500 and 1000 mg/kg doses, suggested that the plant may be acting by facilitating GABAergic transmission. A significant reduction (P< 0.05) in the activity of brain GABA transaminase was observed at higher doses. No neurotoxic signs were observed with rotarod test, pentobarbital induced sleeping time, locomotor activity and haloperidol-induced catalepsy.
The ethanolic extract of tinctoria was found to be useful to control and treat the variety of seizures.
PMCID: PMC3586835  PMID: 23493444
Clonic seizures; GABA; GABA-T; Hind limb tonic extension; Indigofera tinctoria; MES; Neurotoxic; PTZ
16.  Transformation of the flax rust fungus, Melampsora lini: selection via silencing of an avirulence gene 
Rust fungi cause devastating diseases on many important food crops, with a damaging stem rust epidemic currently affecting wheat production in Africa and the Middle East. These parasitic fungi propagate exclusively on plants, precluding the use of many biotechnological tools available for other culturable fungi. In particular the lack of a stable transformation system has been an impediment to the genetic manipulation required for molecular analysis of rust pathogenicity. We have developed an Agrobacterium-mediated genetic transformation procedure for the model flax rust fungus Melampsora lini, which infects flax (Linum usitatissimum). Selection of transgenic rust lines is based on silencing of AvrL567, which encodes a rust effector protein that is recognised by the flax L6 immune receptor. The non-transgenic rust line is unable to infect flax plants expressing L6, while silenced transgenic lines are virulent on these plants, providing an effective selection system. This directly confirms that the cloned AvrL567 gene is responsible for flax rust virulence phenotypes, and demonstrates the utility of this system to probe rust gene function.
PMCID: PMC3142615  PMID: 19874543
rust; transformation; avirulence; effector; gene silencing; Agrobacterium
17.  Phytochemical screening and anticonvulsant studies of ethyl acetate fraction of Globimetula braunii on laboratory animals 
To investigate the phytochemical properties and the anticonvulsant potential of the ethyl acetate soluble fraction of ethanol leaf extract of Globimetula braunii, a plant used in ethnomedicine for the treatment of epilepsy.
The phytochemical screening was carried out using standard protocol while the anticonvulsant activity was studied using maximal electroshock test in chicks, pentylenetetrazole and 4-aminopyridine-induced seizures in mice.
The preliminary phytochemical screening carried out on the crude ethanol extract revealed the presence of saponins, carbohydrates, flavonoids, tannins, anthraquinones and steroids. Similarly, tannins, flavonoids and steroids/terpenes were found to be present in the ethyl acetate fraction. In the pharmacological screening, 150 mg/kg of the fraction protected 83.33% of animals against pentylenetetrazole-induced seizure in mice whereas sodium valproate a standard anti-epileptic drug offered 100% protection. In the 4-aminopyridine-induced seizure model, the fraction produced a significant (P<0.05) increase in the mean onset of seizure in unprotected animals. The fraction did not exhibit a significant activity against maximal electroshock convulsion. The median lethal dose of the fraction was found to be 1 261.91 mg/kg.
These results suggest that the ethyl acetate fraction of Globimetula braunii leaves extract possesses psychoactive compound that may be useful in the management of petit mal epilepsy and lend credence to the ethnomedical use of the plant in the management of epilepsy.
PMCID: PMC3929791
Epilepsy; Globimetula braunii; Seizure; Medicinal; Pentylenetetrazole
18.  Screening of the anticonvulsant activity of some plants from Fabaceae family in experimental seizure models in mice 
Background and purpose of the study
Fabaceae is the third largest family of flowering plants. Lack of essential oils in the plants of this family can be an advantage in search for safe and effective medicines. In this study the anticonvulsant effect of the leaves of Albizzia julibrissin, Acacia juliflora, Acacia nubica and aerial parts of Astragalus obtusifolius was evaluated in pentylenetetrazole (PTZ) and maximal electroshock (MES) seizure tests.
The hydroalcoholic extracts of the plants were obtained by percolation. Different doses of the extracts were injected to the mice intraperitoneally (i.p.) and occurrence of clonic seizures induced by PTZ (60 mg/kg, i.p.) or tonic seizures induced by MES (50 mA, 50Hz, 1sec) were monitored up to 30 min after administration. Acute toxicity of the extracts was also assessed. The safe and effective extract was then fractionated by dichloromethane and anticonvulsant activity of the fractions was determined. Finally, the constituents of the extract and the fractions were screened by thin layer chromatography.
Among the extracts, only A. obtusifolius extract showed low toxicity and protective effect against clonic seizures with ED50 value of 3.97 g/kg. Fractionation of the extract led to increase in anticonvulsant activity and ED50 value of 2.86 g/kg was obtained for the aqueous fraction. Phytochemical screening revealed the presence of alkaloids, flavonoids, anthrones and saponins in the aqueous fraction.
Major conclusion
The presence of anticonvulsant compounds in A. obtusifolius suggests further activity-guided fractionation and analytical studies to find out the potential of this plant as a source of anticonvulsant agent.
PMCID: PMC3304383  PMID: 22615673
Pentylenetetrazole; Maximal electroshock; A. obtusifolius
19.  The antiatherogenic, renal protective and immunomodulatory effects of purslane, pumpkin and flax seeds on hypercholesterolemic rats 
Atherosclerosis remains one of the leading causes of death all over the world. Flax, pumpkin and purslane seeds are rich sources of unsaturated fatty acids, antioxidants and fibers, known to have antiatherogenic activities.
This study was to examine the efficiency of using either flax/pumpkin or purslane/pumpkin seed mixture (components of ω-3 and ω-6) on hyperlipidemia, kidney function and as immunomodulators in rats fed high cholesterol diets.
Materials and Methods:
40 male albino rats were divided into four groups: control group, hypercholesterolemic rats, fed the balanced diet supplemented with cholesterol at a dose level of 2 g/100 g diet; the other two groups of animals fed the same previous hypercholesterolemic diet supplemented with either flax/pumpkin seed mixture or pumpkin/purslane seed mixture at ratio of (5/1) (ω-3 and ω-6).
The present study showed that 2% cholesterol administration caused a significant increase in total cholesterol, total lipids, and triacylglycerol in both serum and liver. Serum phospholipids, LDL-C, and atherogenic index AI also significantly increased compared to control group. Cholesterol-enriched diet significantly increased serum urea, creatinine, sodium and potassium levels as well as significantly increased serum IgG and IgM compared to healthy control. Consumption of flax/pumpkin or purslane/pumpkin seed mixtures by hypercholesterolemic rats resulted in a significantly decrement in lipid parameters and significant improvement in IgG and IgM levels as compared with hypercholesterolemic rats.
Our results suggests that both flax/pumpkin and purslane/pumpkin seed mixtures had anti-atherogenic hypolipidemic and immunmodulator effects which were probably mediated by unsaturated fatty acids (including alpha linolenic acid) present in seed mixture.
PMCID: PMC3271396  PMID: 22362450
Hypercholesterolemia; flax; pumpkin; purslane seeds; kidney; IgG; IgM
20.  Effect of magnesium oxide on the activity of standard anti-epileptic drugs against experimental seizures in rats 
Indian Journal of Pharmacology  2009;41(6):268-272.
To study the effect of oral magnesium oxide supplementation alone and on the activity of standard anti-epileptic drugs in the animal models of maximal electroshock seizures (MES) and chemically (pentylenetetrazole [PTZ])-induced seizures.
Healthy male albino rats were given magnesium oxide (MgO) supplementation orally in various doses (500, 750 and 1000 mg/kg /day) for 4 weeks (day 1 to day 28). On day 0 and day 29, response to MES (180 mA for 0.2 s) was tested 1 h after pre-administration of phenytoin or carbamazepine orally. Similarly, in the other groups, the response to PTZ 40 mg/kg i.p. was tested 1 h after pre-administration of oral sodium valproate.
Oral administration of MgO in a low dose (500 mg/kg) for 4 weeks in healthy rats appears to exert protective effect against MES. High oral doses of MgO (750 and 1000 mg/kg) appear to enhance the activity of phenytoin and carbamazepine in the MES model. MgO supplementation was seen to decrease the latency of PTZ-induced seizures.
The dose of oral MgO appears to have an inverse relation with the protective effect in MES-induced seizure model. High doses of MgO supplementation given orally appear to enhance the activity of standard anti-epileptic drugs in the MES-induced seizure model.
PMCID: PMC2846501  PMID: 20407558
Epilepsy; magnesium; protective
21.  Effect of Shodhana (processing) on Kupeelu (Strychnos nux-vomica Linn.) with special reference to strychnine and brucine content 
Ayu  2011;32(3):402-407.
Kupeelu (Strychnos nux-vomica Linn.) commonly known as nux vomica is a poisonous plant used extensively in various ayurvedic formulations, with great therapeutic significance. Ayurveda recommended the administration of Kupeelu only after purification in different media like cow's urine (Go mutra), cow's milk (Go dugdha), cow's ghee (Go ghrita), Kanji (sour gruel), and so on. Apart from the classical methods some other methods are also adopted by the traditional practitioners using castor oil (Eranda taila), ginger juice (Ardraka swarasa), in the purification of Kupeelu seeds. In the present study an attempt has been made to purify the seeds by performing two different methods (one classical and another traditional) using Kanji and Ardraka swarasa as Shodhana media. This study reveals that both the methods studied reduce the strychnine and brucine contents in comparison to the raw seeds as determined by high performance thin layer chromatography (HPTLC). After purification in Kanji and Ardraka swarasa, the strychnine content was reduced by 39.25% and 67.82%, respectively, and the brucine content in the purified seeds was also found to have decreased by 17.60% and 40.06%, in comparison to the raw seeds.
PMCID: PMC3326892  PMID: 22529660
Ardraka swarasa; brucine; kanji; kupeelu; shodhana; strychnine
22.  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
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
Annals of neurology  2009;65(4):435-447.
Conventional anticonvulsants reduce neuronal excitability through effects on ion channels and synaptic function. Anticonvulsant mechanisms of the ketogenic diet remain incompletely understood. Since carbohydrates are restricted in patients on the ketogenic diet, we evaluated the effects of limiting carbohydrate availability by reducing glycolysis using the glycolytic inhibitor 2-deoxy-D-glucose (2DG) in experimental models of seizures and epilepsy.
Acute anticonvulsant actions of 2DG were assessed in vitro in rat hippocampal slices perfused with 7.5mM [K+]o, 4-aminopyridine (4-AP), or bicuculline and in vivo against seizures evoked by 6 Hz stimulation in mice, audiogenic stimulation in Fring’s mice, and maximal electroshock and subcutaneous Metrazol in rats. Chronic antiepileptic effects of 2DG were evaluated in rats kindled from olfactory bulb or perforant path.
2DG (10mM) reduced interictal epileptiform bursts induced by high [K+]o, 4-AP and bicuculline, and electrographic seizures induced by high [K+]o in CA3 of hippocampus. 2DG reduced seizures evoked by 6 Hz stimulation in mice (ED50 = 79.7 mg/kg) and audiogenic stimulation in Fring’s mice (ED50 = 206.4 mg/kg). 2DG exerted chronic antiepileptic action by increasing afterdischarge thresholds in perforant path (but not olfactory bulb) kindling and caused a 2-fold slowing in progression of kindled seizures at both stimulation sites. 2DG did not protect against maximal electroshock or Metrazol seizures.
The glycolytic inhibitor 2DG exerts acute anticonvulsant and chronic antiepileptic actions and has a novel pattern of effectiveness in preclinical screening models. These results identify metabolic regulation as a potential therapeutic target for seizure suppression and modification of epileptogenesis.
PMCID: PMC2910719  PMID: 19399874
seizures; kindling; glycolysis; 2-deoxy-D-glucose; ketogenic diet; NRSF; CtBP
25.  Sedative and antiepileptic effects of Anthocephalus cadamba Roxb. in mice and rats 
Indian Journal of Pharmacology  2011;43(6):699-702.
The aim of the present study was to evaluate the sedative and antiepileptic activities of ethanolic extract of Anthocephalus cadamba (ACE) bark in various experimental animal models.
Materials and Methods:
ACE was tested at three doses viz. 100, 200 and 400 mg/kg p.o. We used ketamine-induced sleeping time model to test the sedative property of the extract where, onset and duration of sleep were observed. A paradigm of anticonvulsant models (pentylenetetrazole, isoniazid and maximal electroshock-induced seizures) were used to evaluate its protective effect against absence and generalized types of seizures. Onset of clonic convulsions, tonic extension and time of death were observed in PTZ and INH-induced seizure models. In MES model, duration of tonic hind leg extension and onset of stupor were observed.
ACE showed significant increase in ketamine induced sleeping time. It also exhibited significant increase (P<0.05, 0.01 and 0.001) in latency to clonic convulsion, tonic extension and time of death in PTZ and INH models at all tested doses, whereas in the MES model, the lower dose was found to be effective when compared with the higher doses (200 and 400 mg/kg, p.o.).
The results of the present investigation demonstrated that ACE possesses sedative and antiepileptic activities.
PMCID: PMC3229788  PMID: 22144777
Anthocephalus cadamba; isoniazid; maximal electro shock; pentylenetetrazole; sedation; seizures

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