Agrobioinstitute Dragan Tzankov Blvd.

Sofia, Bulgaria

Agrobioinstitute Dragan Tzankov Blvd.

Sofia, Bulgaria
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Kumar M.,University of Manchester | Atanassov I.,University of Manchester | Atanassov I.,Agrobioinstitute Dragan Tzankov Blvd. | Turner S.,University of Manchester
Plant Physiology | Year: 2017

The cellulose synthase complex (CSC) exhibits a 6-fold symmetry and is known as a “rosette.” Each CSC is believed to contain between 18 and 24 CESA proteins that each synthesize an individual glucan chain. These chains form the microfibrils that confer the remarkable structural properties of cellulose. At least three different classes of CESA proteins are essential to form the CSC. However, while organization of the CSC determines microfibril structure, how individual CESA proteins are organized within the CSC remains unclear. Parts of the plant CESA proteins map sufficiently well onto the bacterial CESA (BcsA) structure, indicating that they are likely to share a common catalytic mechanism. However, plant CESA proteins are much larger than the bacterial BcsA protein, prompting the suggestion that these plant-specific regions are important for interactions between CESA proteins and for conferring CESA class specificity. In this study, we have undertaken a comprehensive analysis of well-defined regions of secondary cell wall CESA proteins, with the aim of defining what distinguishes different CESA proteins and hence what determines the specificity of each CESA class. Our results demonstrate that CESA class specificity extends throughout the protein and not just in the highly variable regions. Furthermore, we find that different CESA isoforms vary greatly in their levels of site specificity and this is likely to be determined by the constraints imposed by their position within the CSC rather than their primary structure. © 2017 American Society of Plant Biologists. All rights reserved.

Berkov S.,University of Barcelona | Berkov S.,Agrobioinstitute Dragan Tzankov Blvd | Romani S.,University of Barcelona | Herrera M.,University of Camagüey | And 5 more authors.
Phytotherapy Research | Year: 2011

Crinum zeylanicum is used in folk medicine as a rubefacient in rheumatism, a treatment for malaria or as a poison. Complex alkaloid profiles in C. zeylanicum plant organs were revealed by GC-MS analysis, including several bioactive compounds. Crinine, lycorine, 11-O-acetoxyambelline, ambelline, 6-hydroxybuphanidrine and 6-ethoxybuphanidrine (an artefact of the isolation procedure) were isolated. Crinine, 6-hydroxybuphanidrine and 6-ethoxybuphanidrine showed antiproliferative effects against human tumor cell lines, crinine being the most active (IC 50 14.04μM against HL-60/Dox). The latter compound induced apoptosis in a dose-dependent manner in HL-60 and MDA-MB-231 cell lines. Structure-activity relationships in the studied molecules indicated that the hydrogenation of the double bond at C1-C2 leads to a loss of activity, whereas substitutions at C6, C8 and C11 affect their cytotoxicity. Copyright © 2011 John Wiley & Sons, Ltd.

Berkov S.,Agrobioinstitute Dragan Tzankov Blvd.
Journal of Pharmaceutical and Biomedical Analysis | Year: 2012

Plants of the Amaryllidaceae family are a well-known source of tetrahydroisoquinoline alkaloids with a wide range of biological activities, including antiviral, antitumoral, antiparasitic, psychopharmacological, and acetylcholinesterase inhibitory, among others. Recent advances in the use of GC or LC coupled to MS have allowed a chemically guided isolation of uncommon and bioactive alkaloids. In the present work, analytical methods were applied to study the alkaloid profile of Narcissus broussonetii, a plant endemic to North Africa. Using the GC-MS technique and an in-home mass fragmentation database, twenty-three alkaloids were identified, including the very rare dinitrogenous alkaloids obliquine, plicamine, and secoplicamine. Applying LC-ESI-LTQ-Orbitrap-MS, fragmentation profiles were found to be similar for obliquine and plicamine but different for secoplicamine. Pretazettine, a potent cytotoxic alkaloid, was also isolated from N. broussonetii, although its identification by GC-MS was only possible after a BSTFA-derivatization. The silylated crude methanolic extract only showed the presence of pretazettine-TMS, confirming that tazettine was formed after the alkaloid extraction. The same observation was made in Narcissus cultivars in which tazettine had been detected as the major alkaloid. As part of an ongoing project on MS of Amaryllidaceae alkaloids, the silylated tazettine and pretazettine were studied by GC-MS/MS, and found to differ in their fragmentation routes. Finally, the EtOAc extract of N. broussonetii showed notable in vitro activity against Trypanosoma cruzi, with an IC50 value of 1.77μg/ml. © 2012 Elsevier B.V.

Georgiev V.,Bulgarian Academy of Science | Ivanov I.,Bulgarian Academy of Science | Berkov S.,Agrobioinstitute Dragan Tzankov Blvd. | Pavlov A.,Bulgarian Academy of Science
Acta Physiologiae Plantarum | Year: 2011

The process of alkaloid biosynthesis by Pancratium maritimum shoot culture, cultivated under submerged conditions, was investigated. Twenty-two compounds of different structural types of the Amaryllidaceae alkaloids (tyramine, narciclasine, galanthamine, haemanthamine, lycorine, pancracine, tazettine and homolycorine types) were detected in the studied samples from biomass and cultural liquid. Dominant compounds in the shoots were of tyramine, lycorine and haemanthamine types, whereas in the culture media were found mainly lycorine type compounds. Based on the multi-metabolic estimation of the alkaloid metabolism and physiological peculiarities, liquid cultures of P. maritimum shoots could be defined as prospective biological systems for producing bioactive molecules with acetylcholinesterase inhibitory and apoptotic activities. © 2010 Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków.

Torras-Claveria L.,University of Barcelona | Berkov S.,Agrobioinstitute Dragan Tzankov Blvd. | Codina C.,University of Barcelona | Viladomat F.,University of Barcelona | Bastida J.,University of Barcelona
Industrial Crops and Products | Year: 2014

One hundred and six ornamental varieties of Narcissus were analysed by GC-MS in an attempt to determine their alkaloid profile and the relationships between them were explored by multivariate statistical methods. Leaf and bulb tissues were analysed separately. A total of 61 Amaryllidaceae alkaloids and 2 protoalkaloids were identified and quantified. Different alkaloid profiles were found in leaves and bulbs, with leaves showing a higher quantity and more complex profile of alkaloids. Important differences were also found among plant varieties. k-Means cluster analysis determined five clusters based on similarities in alkaloid composition in both leaves and bulbs. The predominant alkaloids were lycorine-type in leaves, and galanthamine-type in bulbs. A correspondence analysis suggested that a global relationship between horticultural divisions of ornamental Narcissus varieties and k-means clusters based on alkaloid composition was unlikely, although some clusters were found to be particularly associated with certain divisions. © 2014 Elsevier B.V.

Berkov S.,University of Barcelona | Berkov S.,Agrobioinstitute Dragan Tzankov Blvd. | Bastida J.,University of Barcelona | Viladomat F.,University of Barcelona | Codina C.,University of Barcelona
Talanta | Year: 2011

Galanthamine, an acetylcholinesterase inhibitor marketed as a hydrobromide salt for the treatment of Alzheimer's disease, is obtained from some Amaryllidaceae plants. A new method was developed and validated for its quantification by GC-MS in different plant sources: bulbs and leaves from Narcissus confusus; bulbs from N. pseudonarcissus cv. Carlton; and leaves and in vitro cultures from L. aestivum. Samples (50 mg) were extracted with methanol (1 mL) for 2 h, then aliquots of the extracts were silylated and analyzed by GC-MS. The calibration line was linear over a range of 15-800 μg galanthamine/sample, ensuring an analysis of samples with a content of 0.03-1.54% analyte referred to dry weight. The recovery was generally more than 95%. Good inter- and intra assay precision was observed (RSD < 3%). Principal component analysis of GC-MS chromatograms allowed discrimination of the plant raw material with respect to species, organs and geographical regions. The analytical method developed in this study proved to be simple, sensitive and far more informative than the routine analytical methods (GC, HPLC, CE and NMR), so it may be useful for quality control of plant raw materials in the pharmaceutical industry. © 2010 Elsevier B.V. All rights reserved.

Stoykova P.,Agrobioinstitute Dragan Tzankov Blvd | Stoeva-Popova P.,Winthrop University
Plant Cell, Tissue and Organ Culture | Year: 2011

Due to the rising public concern over the use of genes conferring antibiotic and herbicide resistance, alternative systems for selection after plant transformation are being developed. A positive selection system consists of a physiologically inert metabolite as the selection agent and a respective gene which determines a metabolic advantage via selection agent utilization. The transformed cells are able to overcome the suppressive effects of the selection, while the untransformed ones starve but are not killed. The enzyme phosphomannose isomerase (PMI, E. C. 5.3. 1.8) catalyzes the reversible interconversion of mannose-6-phosphate and fructose-6-phosphate in prokaryotic and eukaryotic organisms. The PMI selection system is called positive due to the effect of "starvation" caused to the nontransformed plant tissue because of its incapability to utilize mannose as a carbon source. In this mini-review we researched the literature to obtain a more detailed view of the characteristics, specifics, problems, and advantages of applying the PMI/mannose selection system. © 2010 Springer Science+Business Media B.V.

Berkov S.,University of Barcelona | Berkov S.,Agrobioinstitute Dragan Tzankov Blvd. | Viladomat F.,University of Barcelona | Codina C.,University of Barcelona | And 3 more authors.
Journal of Mass Spectrometry | Year: 2012

Galanthamine-type alkaloids produced by plants of the Amaryllidaceae family are potent acetylcholinesterase inhibitors. One of them, galanthamine, has been marketed as a hydrobromide salt for the treatment of Alzheimer's disease. In the present work, gas chromatography with electron impact mass spectrometry (GC-EIMS) fragmentation of 12 reference compounds isolated from various amaryllidaceous plants and identified by spectroscopic methods (1D and 2D nuclear magnetic resonance, circular dichroism, high-resolution MS (HRMS) and EIMS) was studied by tandem mass spectrometry (GC-MS/MS) and accurate mass measurements (GC-HRMS). The studied compounds showed good peak shape and efficient GC separation with a GC-MS fragmentation pattern similar to that obtained by direct insertion probe. With the exception of galanthamine-N-oxide and N-formylnorgalanthamine, the galanthamine-type compounds showed abundant [M]+. and [M-H]+ ions. A typical fragmentation pattern was also observed, depending on the substituents of the skeleton. Based on the fragmentation pathways of reference compounds, three other galanthamine-type alkaloids, including 3-O-(2′-butenoyl)sanguinine, which possesses a previously unelucidated structure, were identified in Leucojum aestivum ssp. pulchelum, a species endemic to the Balearic islands. GC-MS can be successfully applied to Amaryllidaceae plant samples in the routine screening for potentially new or known bioactive molecules, chemotaxonomy, biodiversity and identification of impurities in pharmaceutical substances. Copyright © 2012 John Wiley & Sons, Ltd.

Torras-Claveria L.,University of Barcelona | Berkov S.,Agrobioinstitute Dragan Tzankov Blvd. | Codina C.,University of Barcelona | Viladomat F.,University of Barcelona | Bastida J.,University of Barcelona
Industrial Crops and Products | Year: 2013

Many plant varieties of Narcissus (Amaryllidaceae) are cultivated on a large scale for ornamental purposes but they are also known to contain galanthamine, a bioactive alkaloid used in antiacetylcholinesterasic therapy in Alzheimer's disease.More than 100 ornamental varieties of Narcissus, using leaves and bulbs separately, were screened for their galanthamine content as well as their acetylcholinesterase inhibitory activity in the search for new potential sources of galanthamine.GC-MS was used for the quantitative analysis of galanthamine, and the biological activity was determined by an acetylcholinesterase in vitro assay.Galanthamine was found to occur in the leaves or bulbs of 97 ornamental varieties, the maximal amount being 0.46% referred to DW in the leaves of Narcissus hispanicus, and 0.14% in the bulbs of the cultivar 'Yellow Wings'. Sanguinine, an even more powerful inhibitor of acetylcholinesterase than galanthamine, was also found in the leaves and bulbs of 22 ornamental varieties of Narcissus, which exhibited a high acetylcholinesterase inhibitory activity. © 2012 Elsevier B.V.

Souza J.M.M.,Federal University of Pará | Berkov S.,Agrobioinstitute Dragan Tzankov Blvd. | Santos A.S.,Federal University of Pará
Anais da Academia Brasileira de Ciencias | Year: 2014

In this study, a protocol to induce high amount of friable callus of Boerhaavia paniculata RICH and a lipidomics technique were applied to investigate the profile of lipids to relate to those present in the roots of this plant that presented anti-inflammatory activity in the crude hexane extract. The callus culture was induced from seeds in solidified Murashige and Skoog medium containing different amounts of glucose and different concentrations of 2,4-Dichlorophenoxyacetic acid . The explants were kept in a germination chamber at 30±2°C with a photoperiod of 16 h under light intensity of 27 μmol m-2 s-1 for 4 weeks. The best results for friable callus formation and development of the biomass were obtained in the treatment containing 2.26 μM 2.4-D and glucose (1.5 %; w/v). Lipidomics techniques were applied in hexane fraction showing higher concentrations of the steroids β-sitosterol (3.53 mg/100 g dc–dry cells), and fatty acids, especially 2-hydroxy-tetracosanoic acid (0.34 mg/100 g dc), eicosanoic acid (86.25 mg/100 g dc), stearic acid (420.83 mg/100 g dc), tetradecanoic acid (10.74 mg/100 g dc) and linoleic acid (100.61 mg/100 g dc). The lipid profile of callus versus that found in the roots of wlid plant is described in this work. © 2014, Academia Brasileira de Ciencias. All rights reserved.

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