Evaluation of free radical quenching, anti-inflammatory activity together with anticancer potential of Lychnis coronaria and characterization of novel molecules from its extract through high resolution-liquid chromatography mass spectrometry coupled to structural biochemistry approach.

Lychnis coronaria, a perennial (herbaceous) belonging to Caryophyllaceae has been traditionally used for treating different complications. However, the free radical scavenging effect, anti-inflammatory activity and anticancer property of methanolic extract of this plant has not been addressed. Most importantly, the chemical constituents present in the extract of Lychnis coronaria responsible for its diverse activities have not been scrutinized till date. Here, we used a complex approach for exploring the above mentioned effects of Lychnis coronaria. We performed rigorous phytochemical screening followed by quantification of tannins, phenols, alkaloids, quinones and sterols from the extract. Moreover we employed in vitro DPPH, ABTS , FRAP assay, albumin denaturation inhibition experiment, MTT assay, high resolution liquid chromatography mass spectrometry for measurng the reactive oxygen species quenching, anti-inflammatory and anticancer strength of Lychnis coronaria and for identifying the possible bioactive molecules. We identified two novel molecules panaxynol (polyacetylenic alcohol) and norharman (9H-Pyrido [3, 4-B] indole) following rigorous analysis of the extract. Following this, the binding affinity of these molecules was estimated using human cyclooxygenase (COX)-2 enzyme as target. Among the constituents of Lychnis coronaria norharman manifested stronger binding towards COX-2 compared to panaxynol. Most importantly, norharman showed high stability in the groove of COX2 as confirmed by molecular dynamics simulation. Collectively, Lychnis coronaria manifested free radical neutralizing, inflammation soothing and anticancer effect in concentration dependent manner and thus may serve as a promising phytotherapeutic in future.Communicated by Ramaswamy H. Sarma.

Development of Colloidal Gold Immunochromatography and Reverse-Transcription Loop-Mediated Isothermal Amplification Assays to Detect Lychnis Mottle Virus.

Lychnis mottle virus (LycMoV; genus Unassigned, family Secoviridae) infection of Angelica sinensis produces mottle and mosaic symptoms, damaging the host. Early detection of relevant pathogens is the most critical step in preventing the potential transmission of infectious disease. Polyclonal antibodies with high potency and high specificity were prepared using the recombinant LycMoV capsid protein as an antigen. Here, we developed and optimized a rapid colloidal gold immunochromatography assay (GICA) detection system for LycMoV using this antibody. Under optimum conditions, GICA specifically detected (up to 10,000-fold) positive LycMoV samples. A real-time reverse-transcription loop-mediated isothermal amplification (RT-LAMP) system was also established by selecting the primers with high sensitivity and specificity to LycMoV. The RT-LAMP detection threshold was 1.42 fg/µl (291 copies/µl). A GICA-RT-LAMP assay system was further established and optimized. The minimum GICA detection line was calculated at 1.52 × 10-2 ng/µl. Although GICA did not detect positive samples after capturing virus at 2.53 × 10-3 ng/µl, GICA-LAMP and GICA-RT-PCR did, whose sensitivity was comparatively greater than sixfold. This is the first report showing that GICA-RT-LAMP is a cost-effective approach for use in detecting LycMoV without extracting nucleic acids. These sensitive assays will help improve virus disease management in A. sinensis crops.

Evaluation of the Gastroprotective Effect of the Flavonoid Complex from Lychnis chalcedonica L. on the Models of Experimental Ulcerogenesis.

Using rat and mouse models of neurogenic, ethanol-induced, and indometacin-induced damage to the gastric mucosa we demonstrated that course preventive treatment with flavonoid complex from aerial parts of Lychnis chalcedonica L. increased the resistance of gastric mucosa to ulcerogenic factors of different etiology. The gastroprotective effect of the phytocomplex in a dose range of 16-1600 µg/kg was comparable with that of the reference drug plantaglucide and was superior to that of the reference drugs eleutherococcus extract and methyluracil in the therapeutic doses. The antiulcerogenic activity of Lychnis chalcedonica flavonoid complex considerably exceeded activity of Lychnis chalcedonica L. extract demonstrated in our previous experiments.

The population structure of the secovirid lychnis mottle virus based on the RNA2 coding sequences.

Lychnis mottle virus (LycMoV), family Secoviridae, is one of several viruses recently detected in peony. Given the high prevalence of the virus in the more than 300 samples tested, the population structure of the virus was studied using 48 isolates representing at least 20 cultivars and collected from major producing and propagating states in the United States. The homogeneity of the United States population, based on data from the RNA2 coding region, along with phylogenetic analyses of all publicly available sequences point to the dissemination of the virus through propagation material rather that active vector-mediated transmission.

Two Ecdysteroids Isolated from Micropropagated Lychnis flos-cuculi and the Biological Activity of Plant Material.

Genetically uniform plant material, derived from Lychnis flos-cuculi propagated in vitro, was used for the isolation of 20-hydroxyecdysone and polypodine B and subjected to an evaluation of the antifungal and antiamoebic activity. The activity of 80% aqueous methanolic extracts, their fractions, and isolated ecdysteroids were studied against pathogenic Acanthamoeba castellani. Additionally, a Microtox® acute toxicity assay was performed. It was found that an 80% methanolic fraction of root extract exerts the most potent amoebicidal activity at IC50 of 0.06 mg/mL at the 3rd day of treatment. Both ecdysteroids show comparable activity at IC50 of 0.07 mg/mL. The acute toxicity of 80% fractions at similar concentrations is significantly higher than that of 40% fractions. Crude extracts exhibited moderate antifungal activity, with a minimum inhibitory concentration (MIC) within the range of 1.25-2.5 mg/mL. To the best of our knowledge, the present report is the first to show the biological activity of L. flos-cuculi in terms of the antifungal and antiamoebic activities and acute toxicity. It is also the first isolation of the main ecdysteroids from L. flos-cuculi micropropagated, ecdysteroid-rich plant material.

Transcriptome-wide mining, characterization, and development of microsatellite markers in Lychnis kiusiana (Caryophyllaceae).

BACKGROUND: Lychnis kiusiana Makino is an endangered perennial herb native to wetland areas in Korea and Japan. Despite its conservational and evolutionary significance, population genetic resources are lacking for this species. Next-generation sequencing has been accepted as a rapid and cost-effective solution for the identification of microsatellite markers in nonmodel plants. RESULTS: Using Illumina HiSeq 2000 sequencing technology, we assembled 67,498,600 reads into 91,900 contigs and identified 11,403 microsatellite repeat motifs in 9563 contigs. A total of 4510 microsatellite-containing transcripts had Gene Ontology (GO) annotations, and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis identified 124 pathways with significant scores. Many microsatellites in the L. kiusiana leaf transcriptome were linked to genes involved in the plant response to light intensity, salt stress, temperature stimulus, and nutrient and water deprivation. A total of 12,486 single-nucleotide polymorphisms (SNPs) were identified on transcripts harboring microsatellites. The analysis of nucleotide substitution rates for 2389 unigenes indicated that 39 genes were under strong positive selection. The primers of 6911 microsatellites were designed, and 40 of 50 selected primer pairs were consistently and successfully amplified from 51 individuals. Twenty-five of these were polymorphic, and the average number of alleles per SSR locus was 6.96, with a range from 2 to 15. The observed and expected heterozygosities ranged from 0.137 to 0.902 and 0.131 to 0.827, respectively, and locus-specific FIS estimates ranged from - 0.116 to 0.290. Eleven of the 25 primer pairs were successfully amplified in three additional species of Lychnis: 56% in L. wilfordii, 64% in L. cognata and 80% in L. fulgens. CONCLUSIONS: The transcriptomic SSR markers of Lychnis kiusiana provide a valuable resource for understanding the population genetics, evolutionary history, and effective conservation management of this species. Furthermore, the identified microsatellite loci linked to the annotated genes should be useful for developing functional markers of L. kiusiana. The developed markers represent a potentially valuable source of transcriptomic SSR markers for population genetic analyses with moderate levels of cross-taxon portability.

Genetic diversity and population structure of Lychnis wilfordii (Caryophyllaceae) with newly developed 17 microsatellite markers.

Lychnis wilfordii (Regel) Maxim. is a perennial plant designated as an endangered species by the Korean government because of rapid reduction in its population size. Thus, a population genetic study of this species is needed to establish the strategy for management and conservation based on scientific evidences. The goals of this study were to develop useful microsatellite markers for L. wilfordii and to understand current genetic status of L. wilfordii in Korean peninsula. Seventeen microsatellite markers were identified using next-generation sequencing and bioinformatic analysis and then analyzed genetic diversity in one hundred forty-five individuals from Korea (KI1, KI2, and KP), China (CX, CF) and Russia (RP). Analysis of molecular variance (AMOVA), principal coordinates analysis (PCoA) and STRUCTURE results consistently showed discontinuity among L. wilfordii populations. AMOVA showed that the percentage of variation among populations was 53%, which was higher than the variation within populations (19%). PCoA showed that the populations were divided into three genetic clusters, (1) Chinese (CX, CF), (2) Russian (RP) populations and Korean populations (KI1, KI2) excluding KP, and (3) the KP population. In particular, KP, the most southern population on the Korean peninsula, showed significantly lower observed and expected heterozygosity, number of effective alleles, and Shannon index (I) than those of KI1 and KI2. L. wilfordii showed high differentiation between populations with low genetic diversity within populations. Among Korean populations, KP is likely to be affected by genetic drift due to small population size, low genetic diversity and limited gene flow.

Construction of infectious clones of lychnis ringspot virus and evaluation of its relationship with barley stripe mosaic virus by reassortment of genomic RNA segments.

Lychnis ringspot virus (LRSV, genus Hordeivirus) was first isolated in 1959, and has been shown to infect several dicot plants in nature. However, due to the lack of infectious cDNA clones, the biological properties and mechanisms underlying LRSV infection are obscure. In this work, we constructed infectious cDNA clones of LRSV and have compiled the complete LRSV genomic (g) RNA sequence. Comparison of nucleotide and amino acid sequences between LRSV and barley stripe mosaic virus (BSMV), the type member of genus Hordeivirus, reveals that despite belonging to the same genus, and replicating in chloroplasts, the viruses are only distantly related. This could be further indicated by the failure of different LRSV/BSMV reassortants to infect N. benthamiana. LRSV infectious cDNA clones provide a useful tool for studies of biological diversity among hordeiviruses, and also may contribute to the understanding of seed transmission in dicot plants.

Anti-Inflammatory and Analgesic Activities of the Complex of Flavonoids from Lychnis chalcedonica L.

Anti-inflammatory and analgesic activities of the complex of flavonoids from Lychnis chalcedonica L. were studied in the models of acute aseptic inflammation induced by carrageenan, histamine, and serotonin and acetic acid-induced painful chemical stimulation. It is demonstrated that course treatment with flavonoids derived from Lychnis chalcedonica L. produced a stable pharmacological effect comparable with that of the reference anti-inflammatory drug diclofenac.

The complete chloroplast genome sequences of Lychnis wilfordii and Silene capitata and comparative analyses with other Caryophyllaceae genomes.

The complete chloroplast genomes of Lychnis wilfordii and Silene capitata were determined and compared with ten previously reported Caryophyllaceae chloroplast genomes. The chloroplast genome sequences of L. wilfordii and S. capitata contain 152,320 bp and 150,224 bp, respectively. The gene contents and orders among 12 Caryophyllaceae species are consistent, but several microstructural changes have occurred. Expansion of the inverted repeat (IR) regions at the large single copy (LSC)/IRb and small single copy (SSC)/IR boundaries led to partial or entire gene duplications. Additionally, rearrangements of the LSC region were caused by gene inversions and/or transpositions. The 18 kb inversions, which occurred three times in different lineages of tribe Sileneae, were thought to be facilitated by the intermolecular duplicated sequences. Sequence analyses of the L. wilfordii and S. capitata genomes revealed 39 and 43 repeats, respectively, including forward, palindromic, and reverse repeats. In addition, a total of 67 and 56 simple sequence repeats were discovered in the L. wilfordii and S. capitata chloroplast genomes, respectively. Finally, we constructed phylogenetic trees of the 12 Caryophyllaceae species and two Amaranthaceae species based on 73 protein-coding genes using both maximum parsimony and likelihood methods.

Colonization and diversification in the African 'sky islands': insights from fossil-calibrated molecular dating of Lychnis (Caryophyllaceae).

The flora on the isolated high African mountains or 'sky islands' is remarkable for its peculiar adaptations, local endemism and striking biogeographical connections to remote parts of the world. Ages of the plant lineages and the timing of their radiations have frequently been debated but remain contentious as there are few estimates based on explicit models and fossil-calibrated molecular clocks. We used the plastid region maturaseK (matK) and a Caryophylloflora paleogenica fossil to infer the age of the genus Lychnis, and constructed a data set of three plastid (matK; a ribosomal protein S16 (rps16); and an intergenic spacer (psbE-petL)) and two nuclear (internal transcribed spacer (ITS) and a region spanning exon 18-24 in the second largest subunit of RNA polymerase II (RPB2)) loci for joint estimation of the species tree and divergence time of the African representatives. The time of divergence of the African high-altitude Lychnis was placed in the late Miocene to early Pliocene. A single speciation event was inferred in the early Pliocene; subsequent speciation took place sporadically from the late Pliocene to the middle Pleistocene. We provide further support for a Eurasian origin of the African 'sky islands' floral elements, which seem to have been recruited via dispersals at different times: some old, as in Lychnis, and others very recent. We show that dispersal and diversification within Africa play an important role in shaping these isolated plant communities.

Genetic variation and selfing rate in Lychnis flos-cuculi along an industrial pollution gradient.

We studied nine populations of a meadow mixed-mating plant Lychnis flos-cuculi growing in a gradient of copper smelter emissions. We hypothesize that metal tolerant populations in the polluted areas have experienced a loss of genetic variation and are more selfing than the populations from the unpolluted areas. One hundred and thirty-five parental plants and 1059 offspring were genotyped with six microsatellite markers. Selfing rates were assessed manually, with Rmes, Mltr and Colony2. Soil toxicity, population density and pollinators' activity were estimated in the studied areas. Populations from the heavily polluted area have experienced a strong founder effect. However, at present, they are characterized by high densities. A recent genetic explosion was registered for the population from the most polluted site, probably due to forest thinning under pollution effects. Selfing rates estimated with different approaches agreed well only for populations with high genetic variation; they comprised 0-0.23 and were similar between polluted and clean areas. Self-fertilization in L. flos-cuculi hardly represents a mechanism for the fixation of advantageous alleles and a barrier for gene flow from non-tolerant populations. The employment of different methods of selfing rate estimation in populations with low genetic variation appears to be necessary, though not a guarantee of reliable conclusions.

The complete nucleotide sequence and genome organization of lychnis mottle virus.

The complete genomic sequence of lychnis mottle virus (LycMoV) from a Lychnis cognata plant was determined. LycMoV has a bipartite genome consisting of RNA1 (7,428 nt) and RNA2 (3,734 nt). Species in the family Secoviridae are demarcated based on their amino acid similarities in the protease-polymerase and coat protein. In LycMoV, these proteins share 90% and 63% sequence similarity, respectively, with the most closely related virus, strawberry latent ringspot virus, which is a member of the family Secoviridae but has not been assigned to a genus. Therefore, LycMoV is a tentative new virus of the family Secoviridae.

Antiulcer activity of extracts of ecdysteroid-containing plants of genera Lychnis and Silene of the Caryophyllaceae family.

We studied antiulcer activity of the extracts of ecdysteroid-containing plants of the Caryophyllaceae family: Lychnis chalcedonica L., Silene viridiflora L.Sp.Pl., and Silene frivaldszkyana Hampe. Experiments on the model of neurogenic and aspirin-induced ulcerogenesis showed unidirectional and pronounced gastroprotective effects of S. viridiflora and L. chalcedonica extracts comparable to the efficacy of famotidine. In these models, a course of intragastric treatment with the extracts reduced ulcerative lesions of all types.

The structural and functional connectivity of the grassland plant Lychnis flos-cuculi.

Understanding the relationship between structural and functional connectivity is essential for successful restoration and conservation management, particularly in intensely managed agricultural landscapes. We evaluated the relationship between structural and functional connectivity of the wetland plant Lychnis flos-cuculi in a fragmented agricultural landscape using landscape genetic and network approaches. First, we studied the effect of structural connectivity, such as geographic distance and various landscape elements (forest, agricultural land, settlements and ditch verges), on gene flow among populations as a measurement of functional connectivity. Second, we examined the effect of structural graph-theoretic connectivity measures on gene flow among populations and on genetic diversity within populations of L. flos-cuculi. Among landscape elements, forests hindered gene flow in L. flos-cuculi, whereas gene flow was independent of geographic distance. Among the structural graph-theoretic connectivity variables, only intrapopulation connectivity, which was based on population size, had a significant positive effect on gene flow, that is, more gene flow took place among larger populations. Unexpectedly, interpopulation connectivity of populations, which takes into account the spatial location and distance among populations, did not influence gene flow in L. flos-cuculi. However, higher observed heterozygosity and lower inbreeding was observed in populations characterised by higher structural interpopulation connectivity. This finding shows that a spatially coherent network of populations is significant for maintaining the genetic diversity of populations. Nevertheless, lack of significant relationships between gene flow and most of the structural connectivity measures suggests that structural connectivity does not necessarily correspond to functional connectivity.

Flavone C-glycosides from Lychnis senno and their antioxidative activity.

A new flavone C-glycoside, 5-O-acetyl-2"-alpha-rhamnopyranosylisovitexin (1), was isolated from the aerial parts of Lychnis senno Siebold et Zucc. (Family Caryophyllaceae), along with 2"-alpha-rhamnopyranosylisovitexin (2), 2"-a-rhamnopyranosylisoorientin (3) and isoorientin 3'-mehyl ether (4). All these compounds were isolated for the first time from L. senno and their antioxidative activity was analyzed by the 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radical-scavenging method. Compound 3 showed moderate antioxidative activity.

Inbreeding alters activities of the stress-related enzymes chitinases and ß-1,3-glucanases.

Pathogenesis-related proteins, chitinases (CHT) and ß-1,3-glucanases (GLU), are stress proteins up-regulated as response to extrinsic environmental stress in plants. It is unknown whether these PR proteins are also influenced by inbreeding, which has been suggested to constitute intrinsic genetic stress, and which is also known to affect the ability of plants to cope with environmental stress. We investigated activities of CHT and GLU in response to inbreeding in plants from 13 Ragged Robin (Lychnis flos-cuculi) populations. We also studied whether activities of these enzymes were associated with levels of herbivore damage and pathogen infection in the populations from which the plants originated. We found an increase in pathogenesis-related protein activity in inbred plants from five out of the 13 investigated populations, which suggests that these proteins may play a role in how plants respond to intrinsic genetic stress brought about by inbreeding in some populations depending on the allele frequencies of loci affecting the expression of CHT and the past levels of inbreeding. More importantly, we found that CHT activities were higher in plants from populations with higher levels of herbivore or pathogen damage, but inbreeding reduced CHT activity in these populations disrupting the increased activities of this resistance-related enzyme in populations where high resistance is beneficial. These results provide novel information on the effects of plant inbreeding on plant-enemy interactions on a biochemical level.

Between-population outbreeding affects plant defence.

Between-population crosses may replenish genetic variation of populations, but may also result in outbreeding depression. Apart from direct effects on plant fitness, these outbreeding effects can also alter plant-herbivore interactions by influencing plant tolerance and resistance to herbivory. We investigated effects of experimental within- and between-population outbreeding on herbivore resistance, tolerance and plant fitness using plants from 13 to 19 Lychnis flos-cuculi populations. We found no evidence for outbreeding depression in resistance reflected by the amount of leaf area consumed. However, herbivore performance was greater when fed on plants from between-population compared to within-population crosses. This can reflect outbreeding depression in resistance and/or outbreeding effects on plant quality for the herbivores. The effects of type of cross on the relationship between herbivore damage and plant fitness varied among populations. This demonstrates how between-population outbreeding effects on tolerance range from outbreeding depression to outbreeding benefits among plant populations. Finally, herbivore damage strengthened the observed outbreeding effects on plant fitness in several populations. These results raise novel considerations on the impact of outbreeding on the joint evolution of resistance and tolerance, and on the evolution of multiple defence strategies.

Possibility for selective accumulation of polyphenolics in tissue cultures of senno (Lychnis senno Siebold et Zucc.).

Senno (Lychnis senno Siebold et Zucc.), a traditional ornamental plant in Japan had been used as a crude drug acting as natural blood thinners. Since tissue culture protocols have been established, we analyzed polyphenol accumulation profiles in shoot culture, multiple shoot culture, and callus culture using the technique of HPLC with a Photodiode Array Detector. By comparing the HPLC profiles at 220-400 nm from extracts of different cultures, 14 putative flavonoids were confirmed as major metabolites in the cultures of senno. Among the 14 compounds detected, 6 were tissue specific metabolites. It appears that the biosynthetic pathway of polyphenolics in Senno is regulated or strongly influenced by how tissues are regenerated and maintained in the in vitro environment. Hence, it may be possible to selectively produce novel secondary metabolites including flavonoids by engineering a target tissue culture procedure developed in the present study.

Effects of experimental inbreeding on herbivore resistance and plant fitness: the role of history of inbreeding, herbivory and abiotic factors.

Inbreeding is common in plant populations and can affect plant fitness and resistance against herbivores. These effects are likely to depend on population history. In a greenhouse experiment with plants from 17 populations of Lychnis flos-cuculi, we studied the effects of experimental inbreeding on resistance and plant fitness. Depending on the levels of past herbivory and abiotic factors at the site of plant origin, we found either inbreeding or outbreeding depression in herbivore resistance. Furthermore, when not damaged experimentally by snail herbivores, plants from populations with higher heterozygosity suffered from inbreeding depression and those from populations with lower heterozygosity suffered from outbreeding depression. These effects of inbreeding and outbreeding were not apparent under experimental snail herbivory. We conclude that inbreeding effects on resistance and plant fitness depend on population history. Moreover, herbivory can mask inbreeding effects on plant fitness. Thus, understanding inbreeding effects on plant fitness requires studying multiple populations and considering population history and biotic interactions.