The Relationship Between Neuroprotective Activity and Antigenotoxic and Acetylcholinesterase Inhibitory Effects of Glaucium corniculatum Extracts

Abstract The purpose of this study was to investigate the relationship between the acetylcholinesterase (AChE) inhibitory and antigenotoxic effect with the neuroprotective activity of Glaucium corniculatum methanol and water extracts rich in rutin and quercetin flavonoids. Neuroprotective activity in terms of cell survival and development against oxidative damage was measured by MTT assay and microscopic analysis in H2O2-induced NGF-differentiated PC12 (dPC12) cells. QRT-PCR and western blot hybridization method was employed for the determination of AChE inhibition of the extracts in the same cell model, and the genotoxic and antigenotoxic effects were identified with Comet assay with human lymphocytes. H2O2-induced vitality loss in dPC12 cells was inhibited in pre-treated cells with these plant extracts. Moreover, extracts stimulated neurite formation and prevented the oxidative stress-induced reduction in neurite growth. In general, it was determined that G. corniculatum methanol extract containing higher amounts of rutin and quercetin was more effective than water extract in terms of AChE inhibitory, antigenotoxic and also neuroprotective effect. In this study, it was shown for the first time that both AChE inhibitory and antigenotoxic effects of G. corniculatum may be effective in neuroprotection and it's protective and therapeutic effects against neurodegeneration may be related to the flavonoid content.

Molecular Identification and Taxonomic Implication of Herbal Species in Genus Corydalis (Papaveraceae).

Many species of Corydalis (Papaveraceae) have been used as medicinal plants in East Asia, and the most well-known species are Corydalis yanhusuo and C. decumbens in the Pharmacopoeia of China. However, authentication of these species remains problematic because of their high morphological variation. Here, we selected 14 closely related species and five genomic regions (chloroplast: matK, trnG, rbcL, psbA-trnH; nuclear: ITS) to explore the utility of DNA barcoding for authenticating these herbs. In addition, the Poisson tree process (PTP) and automatic barcode gap discovery (ABGD) were also used and compared with DNA barcoding. Our results showed that the ITS region was not suitable for molecular analysis because of its heterogeneous nature in Corydalis. In contrast, matK was an ideal region for species identification because all species could be resolved when matK was used along with the other three chloroplast regions. We found that at least five traditional identified species were lumped into one genetic species by ABGD and PTP methods; thus, highlighting the overestimation of species diversity using the morphological approach. In conclusion, our first attempt of molecular analysis of Corydalis herbs presented here successfully resolved the identification of medicinal species and encouraged their taxonomic re-assessment.

Phylogeography of Eomecon chionantha in subtropical China: the dual roles of the Nanling Mountains as a glacial refugium and a dispersal corridor.

BACKGROUND: Mountains have not only provided refuge for species, but also offered dispersal corridors during the Neogene and Quaternary global climate changes. Compared with a plethora of studies on the refuge role of China's mountain ranges, their dispersal corridor role has received little attention in plant phylogeographic studies. Using phylogeographic data of Eomecon chionantha Hance (Papaveraceae), this study explicitly tested whether the Nanling Mountains, which spans from west to east for more than 1000 km in subtropical China, could have functioned as a dispersal corridor during the late Quaternary in addition to a glacial refugium. RESULTS: Our analyses revealed a range-wide lack of phylogeographic structure in E. chionantha across three kinds of molecular markers [two chloroplast intergenic spacers, nuclear ribosomal internal transcribed spacer (nrITS), and six nuclear microsatellite loci]. Demographic inferences based on chloroplast and nrITS sequences indicated that E. chionantha could have experienced a strong postglacial range expansion between 6000 and 1000 years ago. Species distribution modelling showed that the Nanling Mountains and the eastern Yungui Plateau were the glacial refugia of E. chionantha. Reconstruction of dispersal corridors indicated that the Nanling Mountains also have acted as a corridor of population connectivity for E. chionantha during the late Quaternary. CONCLUSIONS: Our results suggest that the Nanling Mountains may acted dual roles as a dispersal corridor in east-west direction and as a glacial refugium in subtropical China during the late Quaternary. The population connectivity mediated by the mountain range and a strong postglacial range expansion are the most likely reasons for the lack of phylogeographic structure in E. chionantha. The hypothesis of dual roles of the mountain range presented here sheds new insights into the phylogeographic patterns of organisms in subtropical China.

Identification of candidate genes involved in isoquinoline alkaloids biosynthesis in Dactylicapnos scandens by transcriptome analysis.

Dactylicapnos scandens (D. Don) Hutch (Papaveraceae) is a well-known traditional Chinese herb used for treatment of hypertension, inflammation, bleeding and pain for centuries. Although the major bioactive components in this herb are considered as isoquinoline alkaloids (IQAs), little is known about molecular basis of their biosynthesis. Here, we carried out transcriptomic analysis of roots, leaves and stems of D. scandens, and obtained a total of 96,741 unigenes. Based on gene expression and phylogenetic relationship, we proposed the biosynthetic pathways of isocorydine, corydine, glaucine and sinomenine, and identified 67 unigenes encoding enzymes potentially involved in biosynthesis of IQAs in D. scandens. High performance liquid chromatography analysis demonstrated that while isocorydine is the most abundant IQA in D. scandens, the last O-methylation biosynthesis step remains unclear. Further enzyme activity assay, for the first time, characterized a gene encoding O- methyltransferase (DsOMT), which catalyzes O-methylation at C7 of (S)-corytuberine to form isocorydine. We also identified candidate transcription factor genes belonging to WRKY and bHLH families that may be involved in the regulation of IQAs biosynthesis. Taken together, we first provided valuable genetic information for D. scandens, shedding light on candidate genes involved in IQA biosynthesis, which will be critical for further gene functional characterization.

[Identification and analysis of Corydalis boweri, Meconopsis horridula and their close related species of the same genus by using ITS2 DNA barcode].

The study is aimed to ensure the quality and safety of medicinal plants by using ITS2 DNA barcode technology to identify Corydalis boweri, Meconopsis horridula and their close related species. The DNA of 13 herb samples including C. boweri and M. horridula from Lhasa of Tibet was extracted, ITS PCR were amplified and sequenced. Both assembled and web downloaded 71 ITS2 sequences were removed of 5. 8S and 28S. Multiple sequence alignment was completed and the intraspecific and interspecific genetic distances were calculated by MEGA 5.0, while the neighbor-joining phylogenetic trees were constructed. We also predicted the ITS2 secondary structure of C. boweri, M. horridula and their close related species. The results showed that ITS2 as DNA barcode was able to identify C. boweri, M. horridula as well as well as their close related species effectively. The established based on ITS2 barcode method provides the regular and safe detection technology for identification of C. boweri, M. horridula and their close related species, adulterants and counterfeits, in order to ensure their quality control, safe medication, reasonable development and utilization.

Evolutionary history of fumitories (subfamily Fumarioideae, Papaveraceae): An old story shaped by the main geological and climatic events in the Northern Hemisphere.

Fumitories (subfamily Fumarioideae, Papaveraceae) represent, by their wide mainly northern temperate distribution (also present in South Africa) a suitable plant group to use as a model system for studying biogeographical links between floristic regions of the Northern Hemisphere and also the Southern Hemisphere Cape region. However, the phylogeny of the entire Fumarioideae subfamily is not totally known. In this work, we infer a molecular phylogeny of Fumarioideae, which we use to interpret the biogeographical patterns in the subfamily and to establish biogeographical links between floristic regions, such as those suggested by its different inter- and intra-continental disjunctions. The tribe Hypecoeae is the sister group of tribe Fumarieae, this latter holding a basal grade of monotypic or few-species genera with bisymmetric flowers, and a core group, Core Fumarieae, of more specious rich genera with zygomorphic flowers. The biogeographical analysis shows a subfamily that originated in East Asia at the end of the Early Cretaceous. From here, ancestral range expansions followed three different directions, one at the beginning of the Late Cretaceous by the ancestor of tribe Hypecoeae towards central Asia, and two during the Cretaceous-Palaeogene transition towards western North America and Indochina by the ancestor of the tribe Fumarieae. The ancestor of Core Fumarieae expanded its range from East Asia into the Himalayas before to the middle Eocene. The uplifts of the Qinghai-Tibetan Plateau together with the zonal climate pattern of the Palaeogene are suggested to be responsible both for the accelerated diversification rate resulting in the origin of the basal lineages of Core Fumarieae as well as for the westward migration of the ancestor of Fumarieae s.str. into the Irano-Turanian region. From here, this latter group reached South Africa during late Eocene and Mediterranean basin during Oligocene. There were two colonization waves of the Mediterranean following two different routes: a northern route during the early Oligocene by the subtribe Sarcocapninae, probably facilitated by the land bridge resulting of the Mediterranean microplate accretion; and a southern route into North Africa, through the Gomphotherium land bridge, taken by the subtribe Fumariinae between late Oligocene and middle Miocene.

Molecular phylogeny of Asian Meconopsis based on nuclear ribosomal and chloroplast DNA sequence data.

The taxonomy and phylogeny of Asian Meconopsis (Himalayan blue poppy) remain largely unresolved. We used the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (nrDNA) and the chloroplast DNA (cpDNA) trnL-F region for phylogenetic reconstruction of Meconopsis and its close relatives Papaver, Roemeria, and Stylomecon. We identified five main clades, which were well-supported in the gene trees reconstructed with the nrDNA ITS and cpDNA trnL-F sequences. We found that 41 species of Asian Meconopsis did not constitute a monophyletic clade, but formed two solid clades (I and V) separated in the phylogenetic tree by three clades (II, III and IV) of Papaver and its allies. Clade V includes only four Asian Meconopsis species, with the remaining 90 percent of Asian species included in clade I. In this core Asian Meconopsis clade, five subclades (Ia-Ie) were recognized in the nrDNA ITS tree. Three species (Meconopsis discigera, M. pinnatifolia, and M. torquata) of subgenus Discogyne were imbedded in subclade Ia, indicating that the present definition of subgenera in Meconopsis should be rejected. These subclades are inconsistent with any series or sections of the present classifications, suggesting that classifications of the genus should be completely revised. Finally, proposals for further revision of the genus Meconopsis were put forward based on molecular, morphological, and biogeographical evidences.

Concerted evolution of satellite DNA in Sarcocapnos: a matter of time.

SarkOne is a genus-specific satellite-DNA family, isolated from the genomes of the species of the genus Sarcocapnos. This satellite DNA is composed of repeats with a consensus length of 855 bp and a mean G+C content of 52.5%. We have sequenced a total of 189 SarkOne monomeric repeats belonging to a total of seven species of the genus Sarcocapnos. The comparative analysis of these sequences both at the intraspecific and the interspecific levels have revealed divergence patterns between species are proportional to between-species divergence according to the phylogeny of the genus. Our study demonstrates that the molecular drive leading to the concerted-evolution pattern of this satellite DNA is a time-dependent process by which new mutations are spreading through genomes and populations at a gradual pace. However, time is a limiting factor in the observation of concerted evolution in some pairwise comparisons. Thus, pairwise comparisons of species sharing a recent common ancestor did not reveal nucleotide sites in transitional stages higher than stage III according to the Strachan's model. By contrast, there was a gradation in the percentage of upper transition stages (IV, V, VI) the more phylogenetically distant the species were. In addition, closely related species shared a high number of polymorphic sites, but these types of sites were not common when comparing more distant species. All these data are discussed in the light of current life-cycle models of satellite-DNA evolution.

[Study on quality control methods for different varieties of Meconopsis of Gansu].

OBJECTIVE: To establish the quantitative determination method and TLC identification of Meconopsis and study the Vis-UV spectrum character for fast identification. METHODS: Meconopsis were identified by TLC, and the content of quercetin was determined by HPLC. RESULTS: The acid hydrolyze condition was HCl-80% MeOH(8:25) 90 degrees C calefaction by water for 1.5 hour. The condition of HPLC: Hypersil-Keystone-C18 (250 mm x 4.6 mm, 5 microm) column, MeOH-Water (50: 50) as mobile phase, detection wavelength at 360nm, flow rate at 1.0 mL/min, column temperature at 35 degrees C, quercetin showed a good linear relationship at a range of 0.01 - 0.12 microg, r =0.9997. The average recovery was 98.83%, and RSD was 1.69% (n = 9). The contents of quercetin in Meconopsis integrifolia and Meconopsis quintuplinervia of Gansu were 0.1135 mg/g and 0.0842 mg/g respectively. CONCLUSION: The method is simple, accurate and specific. It can be used for the quality control and fast identification of Meconopsis.

[Analysis of total alkaloids in Meconopsis quintuplinervia from different localtites of Qinghai].

The contents of total alkaloids in Meconopsis quintuplinervia Regel, grown in the different localtites of Qinghai Province, are detected by the method of spectrophometry. The result showed that total alkaloid in different localities were 0. 0262% to approximately 0.0788% , its mean was 0.0502%. The content of total alkaloids in the herb increased with elevation, not with latitude.

Diversification of CYCLOIDEA-like TCP genes in the basal eudicot families Fumariaceae and Papaveraceae s.str.

CYCLOIDEA-like genes belong to the TCP family of transcriptional regulators and have been shown to control different aspects of shoot development in various angiosperm lineages, including flower monosymmetry in asterids and axillary meristem growth in monocots. Genes related to the CYC gene from ANTIRRHINUM show independent duplications in both asterids and rosids. However, it remains unclear to what extent this affected the evolution of flower symmetry and shoot branching in these and other eudicot lineages. Here, we show that CYC-like genes have also undergone duplications in two related Ranunculales families, Fumariaceae and Papaveraceae s.str. These families exhibit morphological diversity in flower symmetry and inflorescence architecture that is potentially related to functions of CYC-like genes. We present sequences of 14 CYC-related genes covering 9 genera. Phylogenetic analyses indicate the presence of three clades of CYC-like genes. Shared motifs in the region between the TCP and R domains of CYC-like genes between Fumariaceae, Papaveraceae s.str., and AQUILEGIA (Ranunculaceae) indicate that the observed duplications originated from a single CYC gene present in all Ranunculales. RT-PCR expression data suggest that gene duplication and diversification in Fumariaceae and Papaveraceae s.str. was accompanied by divergence in expression patterns.

Phylogenetics of Papaver and related genera based on DNA sequences from ITS nuclear ribosomal DNA and plastid trnL intron and trnL-F intergenic spacers.

BACKGROUND AND AIMS: Representatives from Papaver, Roemeria, Stylomecon and Meconopsis were studied to elucidate phylogenetic relationships between Papaver and these closely allied genera. METHODS: Two molecular data sets were used individually and combined and included sequences from the internally transcribed spacer region (ITS) of 18S-26S nuclear ribosomal DNA and the trnL intron and the trnL-trnF intergenic spacer region of plastid DNA. KEY RESULTS: Parsimony analysis demonstrated that the genus is not monophyletic unless the closely related Roemeria, Stylomecon and Meconopsis cambrica are included in a revised circumscription of Papaver. Three distinct clades are resolved in a combined ITS and trnL-F analysis. Clade 1 consists of Papaver sect. Meconella and Asian Meconopsis. Clade 2 contains a group here identified as Papaver s.s., comprising sections Carinatae, Meconidium, Oxytona, Papaver, Pilosa, Pseudopilosa and Rhoeadium. Clade 3 consists of Papaver sect. Argemonidium and Roemeria refracta. A number of diagnostic indels support these groupings. Within clade 2, sects. Papaver and Rhoeadium are either not monophyletic or lack evidence supporting their monophyly. CONCLUSIONS: The results of this molecular analysis indicate that a number of morphological characters such as valvate capsule dehiscence, dark or light filaments and sessile stigmatic discs have arisen in parallel. The phylogenetic trees are incongruent with the existing taxonomy of Papaver, and a revised classification is suggested.