UHPLC-TOFMS coupled with chemometric method as a powerful technique for rapid exploring of differentiating components between two Ziziphus species

To rapidly explore the differentiating components and the potential chemical markers for discrimination between those Chinese medicinal herbs with similar chemical characteristics, an ultra-high-performance liquid chromatography (UHPLC)-TOFMS coupled with multivariate statistical analysis method was proposed and validated by using two Ziziphus species (Z. jujuba and Z. jujuba var. spinosa) as the model herbs. After the samples were analyzed using UHPLC-TOFMS, the data sets of retention time (RT)-m/z pairs, ion intensities and sample codes were further processed with orthogonal partial least squared discriminant analysis (OPLS-DA) to holistically compare the difference between the fruits of these two Ziziphus species, and to generate an S-plot. Those compounds correlating to the points at the two ends of "S" were regarded as the most differentiating components between these two kinds of samples. By comparing the mass/UV spectra and retention times with those of reference compounds, these components were finally characterized as zizyberenalic acid, palmitoleic acid, oleic acid, pomonic acid and rutin, and these compounds would be the potential chemical markers for discrimination of these jujube products. The results suggested that this newly established approach could be used to rapidly determine the subtle differences and explore the potential chemical markers for differentiation within the herbs with similar chemical ingredients.     

A novel method for the analysis of 20 multi‐Indel polymorphisms and its forensic application

Insertion/deletion polymorphisms (Indels) have been considered as potential markers for forensic DNA analysis. However, the discrimination power of Indels is relatively lower due to the poor polymorphisms of diallelic markers. Here, two to three Indel loci that were very tightly linked in physical position were combined into a specific multi‐Indel marker to improve the discrimination, as well as a multiplex that consisted of a set of multi‐Indel markers was developed for forensic purpose. Finally, a multiplex system with 20 multi‐Indel markers including 43 Indel loci from dbSNP database was constructed and DNA sample can be analyzed by this multiplex in one PCR reaction and one CE run. A total of 150 unrelated individuals from Hunan province in South‐central China were genotyped by the multiplex system. The result showed that a total of 63 specific amplicons were detected, three alleles were observed in multi‐Indel markers including two Indel loci and four alleles were observed in the markers including three Indel loci. The cumulative probability of exclusion and the accumulated discrimination power were 0.9989 and 0.9999999999994, respectively. Our result demonstrated that the strategy could be efficient to develop higher polymorphic multi‐Indel markers, and the new multiplex could provide Supporting Information for forensic application.     

Indole alkaloids from Vinca major and V. minor growing in Turkey

A new indole alkaloid, 11-hydroxypolyneuridine, was isolated from Vinca major subsp. major L. and the known indole alkaloids vallesiachotamine and isovallesiachotamine from Vinca minor L. This is the first report on the alkaloids of both Vinca species growing in Turkey; vallesiachotamine and isovallesiachotamine were isolated from a Vinca species for the first time. V. minor may be considered as a new source for these two alkaloids due to their occurrence in high amount in the aerial parts of the plant. The alkaloid extracts of the two Vinca species were found to have high lipid peroxidation inhibitory and DPPH radical scavenging activities. Anticholinesterase activity of the extracts was also very strong.     

Association between chemical and genetic variation of Vitex rotundifolia populations from different locations in China: its implication for quality control of medicinal plants

Vitex rotundifolia is a widely distributed plant species that has been extensively used in traditional Chinese medicine. Its fruits, Fructus Viticis, are recorded as Manjingzi in the Pharmacopoeia of the People's Republic of China. For the effective quality control of its medicinal values reflected by chemical variation patterns, in addition to the relationship with genetic diversity, analyses based on high-performance liquid chromatographic (HPLC) fingerprinting and inter simple sequence repeat (ISSR) molecular markers were carried out, involving 14 V. rotundifolia populations from different locations in China. The HPLC data showed considerable variation of chemical constituents among the V. rotundifolia populations. The hierarchical clustering analysis further revealed four major groups based on their chemotype variation. Abundant genetic diversity was detected among the V. rotundifolia populations that also were clustered into four groups based on their ISSR data. It is important to point out that the genetic variation pattern revealed by molecular markers was closely associated with that indicated by chemical constitutes in the fruits of V. rotundifolia. This finding provides a solid basis for the combined use of chemical and genetic fingerprints in efficiently evaluating qualities and choosing favourable chemotypes with appropriate pharmacological properties of V. rotundifolia, in addition to establishing good agricultural practices for medicinal plants.     

Analysis of DNA restriction fragments and polymerase chain reaction products by capillary electrophoresis

Capillary electrophoresis (CE) is a new, high-resolution tool for the analysis of DNA restriction fragments and DNA amplified by the polymerase chain reaction (PCR). By combining many of the principles of traditional slab gel methods in a capillary format, it is possible to perform molecular size determinations of human and plant PCR amplification products and DNA restriction fragments. DNA restriction fragments and PCR products were analyzed by dynamic sieving electrophoresis (DSE) and capillary gel electrophoresis (CGE). As part of this study, sample preparation procedures, injection modes, and the use of molecular mass markers were evaluated. Optimum separations were performed using the uPage-3 (3% T, 3% C) CGE columns with UV detection at 260 nm. Membrane dialysis and ultrafiltration/centrifugation proved to be nearly equivalent methods of sample preparation. Reproducibility studies demonstrated that blunt-ended, non-phosphorylated markers (specifically allele generated markers) provide the most accurate calibration for PCR product analysis. This study demonstrates that CE offers a high-speed, high-resolution analytical method for accurately determining molecular size and/or allelic type as compared with traditional methodologies.     

Interaction of oxo-bridged vanadium(III) phenanthroline and bipyridine dimers with DNA

Cationic mu-oxo V(III) dimers of the type [V2OL4Cl2]2+ (L = 1,10-phenanthroline, 3,4,7,8-tetramethyl-1,10-phenanthroline, 4,7-diphenyl-1,10-phenanthroline; or 2,2'-bipyridine) are shown to interact very strongly with DNA and to lead ultimately to its degradation. Spectroscopic binding studies, electrophoreses, DNA melting temperature experiments, and other tests on the parent 1,10-phenthroline complex all yield results consistent with tight binding. However, the exact nature of the binding--i.e., intercalative, groove binding, electrostatic, or covalent--remains unclear. Resonance Raman spectroscopy is found to be a powerful method for studying the interaction of these mu-oxo V(III) dimers with DNA and shows that in frozen aqueous solution, the parent complex [V2O(phen)4Cl2]2+ undergoes initial aquation, followed by the reaction of the aquated species with the DNA. Once the V(III) dimer is bound to the DNA, redox takes place, leading to the formation of alkaline-sensitive lesions. Hydrogen peroxide is implicated as a partner in this redox event, based on the effects of the enzymes SOD and catalase.     

Identification and quantification of different species in animal fibres by LC/ESI‐MS analysis of keratin‐derived proteolytic peptides

In the present paper, a proteomic method for species determination in fibres has been developed. Keratin was extracted from yak, wool and cashmere fibres and digested by trypsin, providing peptide mixtures that were analyzed by liquid chromatography coupled with electrospray mass spectrometry (LC/ESI‐MS) in order to identify peptidic species‐specific markers able to differentiate the fibres. Several suitable peptide markers were identified and validated in different fibres of different origin and having undergone different technological treatments, showing 100% specificity and 100% selectivity. Most of the peptide markers were also identified by means of high‐resolution mass spectrometry, confirming the origin from species‐specific keratin sequences. Some peptides were also used for the quantification of the different species in mixed fibres by LC/ESI‐MS. Validation experiments and blind tests confirmed their ability to act as very specific quantitative and qualitative markers. The method here developed is a valid complement to the standard benchmark methods for fibre identification and quantification and will be very useful for assessing the authenticity of textile products. Copyright © 2013 John Wiley & Sons, Ltd.     

Chemosystematics of Porella (Marchantiophyta, Porellaceae)

The taxonomy of the liverwort genus Porella based on plant morphology has been regarded as difficult. Recent DNA-based studies have brought new insights into the systematics of these liverworts and have uncovered some novel relationships that allowed the resolution of controversial treatments based on morphology. One of the outstanding features of these plants, in addition to their form, is their chemical composition, which is characterized by great diversity of secondary metabolites. In this paper the sesqui- and diterpenoids occurring in Porella species are described and their chemosystematic relevance is explored. On the basis of chemical data, the Porella species have been divided into six chemotypes: the drimane- (I), sacculatane- (II), pinguisane-sacculatane- (III), guaiane-germacrane- (IV), pinguisane- (V) and africane- (VI) types. Species belonging to type I are characterized by their hot taste, whereas the other chemotypes are comprised of non-pungent species. Consideration of recent DNA data shows striking correlations between molecular groups and their terpenoid chemistry. The chemical data suggest that the P. vernicosa complex (chemotype I) deserves recognition as a separate section of Porella and that terpenoids are important chemosystematic markers in the family Porellaceae.     

Enantioselective DNA threading dynamics by phenazine-linked.

The interactions between the stereoisomers of the chiral bis-intercalator [mu-C4(cpdppz)(2)-(phen)(4)Ru(2)](4+) and DNA reveal interesting dynamic discrimination properties. The two enantiomers Delta-Delta and Lambda-Lambda both form very strong complexes with calf thymus DNA with similar thermodynamic affinities. By contrast, they display considerable variations in their binding kinetics. The Delta-Delta enantiomer has higher affinity for calf thymus DNA than for [poly(dA-dT)](2), and the association kinetics of the dimer to DNA, as well as to polynucleotides, requires a multiexponential fitting function. The dissociation reaction, on the other hand, could be described by a single exponential for [poly(dA-dT)](2), whereas two exponentials were required for mixed-sequence DNA. To understand the key mechanistic steps of the reaction, the kinetics was studied at varied salt concentration for different choices of DNA and chirality of the threading complex. The enantiomers were found to have markedly different dissociation rates, the Lambda-Lambda enantiomer dissociating about an order of magnitude faster than the Delta-Delta enantiomer. Also, the salt dependence of the dissociation rate constants differed between the enantiomers, being stronger for the Lambda-Lambda enantiomer than for the Delta-Delta enantiomer. Since the dissociation reaction requires unthreading of bulky parts of the bis-intercalator through the DNA helix, a considerable conformational change of the DNA must be involved, possibly defining the rate-limiting step.     

Studies on the genetic variation of the green unicellular alga Haematococcus pluvialis (Chlorophyceae) obtained from different geographical locations using ISSR and RAPD molecular marker

Haematococcus pluvialis (Flotow) is a unicellular green alga, which is considered to be the best astaxanthin-producing organism. Molecular markers are suitable tools for the purpose of finding out genetic variations in organisms; however there have been no studies conducted on ISSR or RAPD molecular markers for this organism. The DNA of 10 different strains of H. pluvialis (four strains from Iran, two strains from Finland, one strain from Switzerland and three strains from the USA) was extracted. A genetic similarity study was carried out using 14 ISSR and 12 RAPD primers. Moreover, the molecular weights of the bands produced ranged from 0.14 to 3.4 Kb. The PCA and dendrogram clustered the H. pluvialis strains into various groups according to their geographical origin. The lowest genetic similarity was between the Iran2 and USA2 strains (0.08) and the highest genetic similarity was between Finland1 and Finland2 (0.64). The maximum numbers of bands produced by the ISSR and RAPD primers were 35 and 6 bands, respectively. The results showed that ISSR and RAPD markers are useful for genetic diversity studies of Haematococcus as they showed geographical discrimination.     

Development and Characterization of Microsatellite Markers (SSR) in Sesamum (Sesamum indicum L.) Species

Microsatellites, also known as simple sequence repeats (SSRs), are the class of repetitive DNA sequences present throughout the genome of many plant and animal species. Recent advances in molecular genetics had been the introduction of microsatellite markers to investigate the genetic structuring of natural plant populations. We have employed an enrichment strategy for microsatellite isolation by using multi-enzymes digestion, microsatellite oligoprobes, and streptavidin magnetic beads in Sesamum (Sesamum indicum L.). More than 200 SSR motifs were detected (SSR motifs ??2 repeat units or 6?bp); 80?% of the clones contained SSR motifs. When regarding SSRs with four or more repeat units and a minimum length of 10?bp, 132 of them showed repeats. Eighteen SSR markers were initially characterized for optimum annealing temperature using a gradient PCR technique. Among the 18 SSR markers characterized, five were found to be polymorphic and used to analyze 60 Sesamum germplasm accessions. The maximum number of alleles detected was four with a single primer and the least number of two alleles with three primers with an average PIC value of 0.77. SSRs are a valuable tool for estimating genetic diversity and analyzing the evolutionary and historical development of cultivars at the genomic level in sesame breeding programs.     

Chromatographic Fingerprint Analysis of Varietal Differences among Three Species of Baishouwu and Simultaneous Analysis of Three Bioactive Constituents by Use of LC–DAD

For the first time, a validated high-performance liquid chromatographic method with diode-array detection has been developed for discrimination and quality assessment of three species of Baishouwu. LC with gradient elution was performed on extracts from 23 plant samples collected from different geographic locations. The fingerprint was established and 26 chromatographic peaks were selected as characteristic peaks. Similarities of chromatograms were greater than 0.9 for the same species but much lower than 0.6 for different species. Three bioactive constituents were simultaneously analyzed for further quantitative assessment of the quality of Baishouwu, and amounts of the three analytes in different species of Baishouwu samples were found to be significantly different. This LC–DAD method is thus a very reliable and useful method for assessment of the quality of Baishouwu.     

Plant DNA fingerprinting with radioactive and digoxigenated oligonucleotide probes complementary to simple repetitive DNA sequences

The existence of hypervariable DNA sequences in nuclear genomes, and the use of appropriate "fingerprinting" probes to detect them, has gained widespread scientific interest, and also led to multiple applications in diverse areas. Two years ago, the new technique of "DNA fingerprinting" was also introduced into the analysis and characterization of plant genomes, initially by using human or M13 minisatellites as probes. In the present article, we demonstrate the applicability for plant DNA fingerprinting of oligonucleotide probes specific for simple repetitive DNA sequences. We show that various levels of intra- and interspecific polymorphisms can be detected; the information to be gained depends on the optimal combination of probe and species. Variety-specific patterns were obtained in several cases. Some probes revealed variability between individuals. Somatic variability was not observed. Different DNA isolation and purification procedures were tested in order to introduce a fast and easy-to-perform isolation method suitable for a large variety of plant species. Nonradioactive fingerprinting was performed using digoxigenated oligonucleotides as probes. Banding patterns obtained with radioactive and digoxigenin-based labeling techniques proved to be of similar quality.     

Polyionic charge density plays a key role in differential recognition of mobile ions by biopolymers

We address the question of what are the molecular mechanisms providing discrimination between seemingly similar counterions binding to various biomolecular surfaces. In the case of protein association with Na (+) and K (+) ions, recent works proposed that specificity of carboxylate functional groups interacting with these mobile ions rationalizes the observed ionic discrimination. We probe in this work whether similar arguments may be used to explain higher propensity of Na (+) ions to associate with DNA compared with K (+) ions, which was suggested by our simulations and some experiments. By comparing our extensive molecular dynamics simulations of Na (+) and K (+) distributions around a 16-base-pair DNA oligomer, [(CGAGGTTTAAACCTCG)] 2, with additional simulations where DNA is replaced by a "soup" of monomers (dimethylphosphate anion), we conclude that DNA specificity toward Na (+)/K (+) is not determined by the underlying functional group specificity. Instead, the collective effect of DNA charges drives larger Na (+) association. To gain additional microscopic insights into the mechanisms of specificity on ionic associations in these systems, we carried out energetic analysis of the association between Na (+) and K (+) with chloride and dimethylphosphate anions. The insights gained from our computational work shed light on a number of experiments on electrolyte solutions of monovalent salts and DNA.     

Locked nucleic acid molecular beacons

A novel LNA-MB (molecular beacon based on locked nucleic acid bases) has been designed and investigated. It exhibits very high melting temperature and is thermally stable, shows superior single base mismatch discrimination capability, and is stable against digestion by nuclease and has no binding with single-stranded DNA binding proteins. The LNA-MB will be widely useful in a variety of areas, especially for in vivo hybridization studies.     

Comparative study of Puerariae lobatae and Puerariae thomsonii by HPLC-diode array detection-flow injection-chemiluminescence coupled with HPLC-electrospray ionization-MS

An on-line HPLC-diode array detection-flow injection chemiluminescence (HPLC-DAD-FICL) method was applied to estimate the difference of Puerariae lobatae and Puerariae thomsonii. Their chemical and active profiles could be obtained by HPLC-DAD-FICL in one run. Seventeen compounds in two species were tentatively identified by HPLC-electrospray ionization-MS (HPLC-ESI-MS) method. The main antioxidants were rapidly screened by active fingerprints coupled with MS data. Similarity and Hierarchical clustering analysis (HCA) were used to distinguish different samples. The results suggested that the chemical fingerprints of 16 batches of samples were similar by similarity evaluation, while HCA could discriminate the two species. The active fingerprints of Puerariae lobatae and Puerariae thomsonii were significantly different. More antioxidants were found in Puerariae lobatae than in Puerariae thomsonii. Main antioxidants, including 3'-hydroxypuerarin, genistein 8-C-glycoside-xyloside, puerarin, 6″-O-xylosylpuerarin, mirificin and daidzein in two species, may be reasonable markers for the discrimination of the two species. The integrated fingerprint based on the chemical and active characteristics may provide an objective quality evaluation for Puerariae lobatae and Puerariae thomsonii.     

Synthetic mimicking of plant oils and comparison with naturally grown products in polyurethane synthesis

The use of plant oils as industrial feedstocks can often be hampered by their lack of optimization towards a particular process, as well as their development being risky; growing suitable volumes of crops to test can take up to five years. To circumvent this, we aimed to discover a method that would mimic plant oil profiles in the laboratory, and show that they exhibited similar properties to the naturally grown plant oils in a given process. Using the synthesis of polyurethanes as an example, we have synthesized six different polymers and demonstrated that plant oils will produce polymers with similar physical properties to those oils mimicked in the laboratory. The use of this mimicking process can be extended to other types of polymers to obtain a method for predicting the properties of a given material based on the plant oil composition of a crop before it is grown in bulk.     

Identification and cloning of molecular markers for UV-B tolerant gene in wild sugarcane (Saccharum spontaneum L.)

Previously we have selected wild sugarcane (Saccharum spontaneum L.) sterile lines that are tolerant or susceptible to UV-B radiation based on response index (RI) in a field screening test. The RI was established according to plant height, tiller number, leaf index, total biomass and brix under enhanced ultraviolet-B (UV-B, 280-310 nm) radiation. In this experiment, molecular markers linked to the UV-B tolerant and susceptible genes were identified and cloned. RAPD (Randomly amplified polymorphic DNAs) assay using 100 arbitrary primers followed by clustering analysis separated the tolerant and susceptible lines into two groups at the genetic distance of 0.380. The UV-B tolerant and susceptible gene pools were constructed and compared using the Bulked Segregate Analysis (BSA) approach. Of the 100 arbitrary RAPD primers, primer OPR16 produced polymorphic DNA banding patterns from both gene pools. The OPR16-1200 bp DNA fragment was only amplified from the tolerant lines and the OPR16-800 bp from the susceptible ones. These two PCR fragments were cloned onto T-vector. DNA sequence alignment analysis determined that 42% homology existed between the reverse and forward sequences of the OPR16-1200 bp clone, and 36% homology between the forward sequences of the OPR16-800 bp and OPR16-1200 bp clones. The two DNA clones were determined to be linked to the UV-B tolerant and susceptible genes, and they can be used to develop molecular markers for the associated traits.     

Simultaneous determination of inorganic and organic antimony species by using anion exchange phases for HPLC-ICP-MS and their application to plant extracts of Pteris vittata

Antimony is a common contaminant at abandoned sites for non-ferrous ore mining and processing. Because of the possible risk of antimony by transfer to plants growing on contaminated sites, it is of importance to analyze antimony and its species in such biota. A method based on high performance liquid chromatographic separation and inductively coupled plasma mass spectrometric detection (HPLC-ICP-MS) was developed to determine inorganic antimony species such as Sb(III) and Sb(V) as well as possible antimony-organic metabolisation products of the antimony transferred into plant material within one chromatographic run. The separation is performed using anion chromatography on a strong anion exchange column (IonPac AS15/AG 15). Based on isocratic optimizations for the separation of Sb(III) and Sb(V) as well as Sb(V) and trimenthylated Sb(V) (TMSb(V)), a chromatographic method with an eluent gradient was developed. The suggested analytical method was applied to aqueous extracts of Chinese break fern Pteris vittata samples. The transfer of antimony from spiked soil composites into the fern, which is known as a hyperaccumulator for arsenic, was investigated under greenhouse conditions. Remarkable amounts of antimony were transferred into roots and leaves of P. vittata growing on spiked soil composites. Generally, P. vittata accumulates not only arsenic (as shown in a multiplicity of studies in the last decade), but also antimony to a lower extent. The main contaminant in the extracts was Sb(V), but also elevated concentrations of Sb(III) and TMSb(V) (all in μg L⁻¹ range). An unidentified Sb compound in the plant extracts was detected, which slightly differ in elution time from TMSb(V).     

RP-HPLC analysis of phenolic acids of selected Central European Carex L. (Cyperaceae) species and its implication for taxonomy

Eighteen species belonging to the Carex genus were checked for the presence and the amount of eight phenolic acids (p-hydroxybenzoic, vanillic, caffeic, syringic, protocatechuic, p-coumaric, sinapic, and ferulic) by means of HPLC. Both the free and bonded phenolic acids were analyzed. The majority of the analyzed acids occurred in the studied species in relatively high amounts. The highest concentrations found were caffeic acid and p-coumaric acid, for which the detected levels were negatively correlated. A very interesting feature was the occurrence of sinapic acid, a compound very rarely detected in plant tissues. Its distribution across the analyzed set of species can be hypothetically connected with the humidity of plants' habitats. Several attempted tests of aggregative cluster analysis showed no similarity to the real taxonomical structure of the genus Carex. Thus, the phenolic acids' composition cannot be considered as the major taxonomical feature for the genus Carex.