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- Recovery of high purity plumbagin from Drosera intermediaPublication . Grevenstuk, Tomás; Gonçalves, Sandra; Nogueira, José Manuel F.; Bernardo-Gil, Maria Gabriela; Romano, AnabelaIn this study a simple process encompassing an efficient extraction and fractionation method to obtain high purity plumbagin from micropropagated D. intermedia plants is described. Plumbagin is a naphthoquinone derived from a diverse but restricted group of plant species that includes the Drosera spp. and is in demand for pharmacological research. It was brought initially to the attention of researchers due to its broad antimicrobial and antitumor properties but has continued to find application against new pharmacological targets. The procedure described in this work involved testing four extraction methods to maximize product recovery and separating the 30–50% acetonitrile in water fraction (v/v) on a SPE column followed by lyophilisation. By applying ultrasonic treatment to the plant matrix leached in nhexane followed by a single step purification process, 2.74 mg of plumbagin per gram of plant material could be obtained with a recovery of 86.3% and over 99% purity.
- Micropropagation for the production of high quality phytochemicalsPublication . Grevenstuk, Tomás; Romano, AnabelaPlants area source of many valuable secondary metabolites that find a broad field of applications, ranging from the agrochemical to the pharmaceutical industries. Establishing a suitable source for extraction of phytochemicals is, however, not always straightforward. In many instances the production by chemical synthesis is not economically viable due to their complex structures and conservation issues may arise when they are harvested from natural sources. In vitro culture techniques offer an attractive alternative to these issues. Natural grown plants can be replaced by in vitro produced biomass with the advantage that several strategies can be implemented to increase production yields, such as genotype selection, altering growth conditions and use of elicitors, so that the higher investment costs are justified. Also, because plant tissue cultures can be generated on a continuous year-round basis without seasonal constraints, they can guarantee reliable and predictable production levels, which is of great importance for efficient process down-stream. Plant tissue culture techniques offer the possibility of establishing cultures from leaves, stems, roots and meristems, meaning that metabolites produced in specific plant organs can also be prospected. The successful production of a large number of phytochemicals from micropropagated biomass has been reported, and it seems that only in a few cases cultures fail to accumulate compounds of interest. The advantages and the range of possibilities offered by plant tissue culture techniques suggest that these might become a valuable and indispensable tool for the production of phytochemicals. In this work, the example of the prospection of plumbagin from micropropagated D. intermedia plants is described. Plumbagin is a naphthoquinone with potential pharmaceutical applications and results obtained by several hyphenated analytical techniques confirm that an end product with high purity and recoveries can be obtained from in vitro cultured plants. © ISHS 2013.
- Aluminium speciation and internal detoxification mechanisms in plants: where do we stand?Publication . Grevenstuk, Tomás; Romano, AnabelaAluminium (Al) typically has a deleterious impact on plant growth and development but some plants can accumulate otherwise toxic concentrations of this metal by deploying internal detoxification mechanisms (tolerance mechanisms). Although there are scattered reports concerning the regulation of Al uptake, transport and storage in metabolically innocuous forms, the overall picture remains incomplete and there are no accounts of a general overview of Al tolerance mechanisms. This review aims to compile the current knowledge on the physiological basis of Al tolerance and speculate on possible underlying molecular mechanisms regarding the uptake of Al, speciation in internal organs, transport to upper organs and storage in non-vital structures. Given the difficulties in detecting and studying the speciation of Al, special attention will be given to the approaches used so far and new developments that allow greater sensitivity and accuracy in uncovering the fate of Al in planta. © 2013 The Royal Society of Chemistry.
- Antioxidant and anti-cholinesterase activities of Lavandula viridis L’Hér extracts after in vitro gastrointestinal digestionPublication . Costa, Patrícia; Grevenstuk, Tomás; Costa, Ana M. Rosa da; Gonçalves, Sandra; Romano, AnabelaLavandula viridis L’Hér is an aromatic shrub with relevant biological activities associated to the presence ofphenolic compounds. However, these compounds must be bioavailable to exert their biological function.Therefore, in this study, we investigated the influence of the L. viridis extract matrices on the digestion ofthe main component rosmarinic acid and their antioxidant and anti-cholinesterase activities after in vitrogastrointestinal digestion. Overall, the antioxidant and anti-cholinesterase activities were assured afterin vitro gastrointestinal processes and we observed that the L. viridis extract matrices have an importantrole in the bioactive effects of their main compound, rosmarinic acid. The L. viridis extracts and rosmarinicacid did not show any toxic effect on colon adenocarcinoma Caco-2 cell viability. Finally, the compoundsfrom L. viridis extracts were not metabolized by Caco-2 cells and were not able to permeate into them.
- Phytochemical studies and biological activity of carnivorous plants from the Mediterranean regionPublication . Grevenstuk, Tomás; Romano, AnabelaIn this thesis several studies were conducted with four carnivorous plant species which occur on Portuguese territory: Pinguicula lusitanica, Pinguicula vulgaris, Drosera intermedia and Drosera rotundifolia. Most habitats of these plants are threatened and natural populations are scarce, therefore micropropagation protocols were developed to produce biomass for the subsequent studies. Efficient micropropagation protocols were developed for P. lusitanica and D. intermedia enabling large scale biomass production, while protocols for the other two species have still to be optimized (in Chapter 2). The in vitro established cultures represent active germplasm collections of Portuguese natural populations and contribute therefore for their conservation. In Chapter 3 extracts prepared from micropropagated plant material were analyzed using state of the art HPLC-ESI-MS and HPLC-SPE-NMR equipment which enabled the identification of the major secondary metabolites produced by P. lusitanica and D. intermedia, directly from essentially crude extracts. The metabolites identified in P. lusitanica belong to the iridoid glucosides and caffeoyl phenylethanoid glycosides and D. intermedia was shown to produce mainly flavonoid glucosides, ellagic acid derivatives and the naphthoquinone plumbagin. The evaluation of the biological activities of these extracts, compiled in Chapter 4, showed that the methanol extract of P. lusitanica has considerable antioxidant activity and that the n-hexane extract of D. intermedia has high antimicrobial potential. In Chapter 5 a method for the extraction of plumbagin from micropropagated D. intermedia plants was optimized and its potential as an alternative for bioprospection evaluated. It was shown that the commercial exploitation of plumbagin from D. intermedia cultures might be viable and that UAE with n-hexane followed by an SPE purification step is an efficient procedure for obtaining large quantities of high purity plumbagin. It is hoped that this study represents an enrichment of the knowledge on these plants and contributes to their conservation and valorisation.
- Inhibitory activity of plumbagin produced by Drosera intermedia on food spoilage fungiPublication . Grevenstuk, Tomás; Gonçalves, Sandra; Domingos, Telma; Quintas, Célia; van der Hooft, Justin J. J.; Vervoort, Jacques; Romano, AnabelaBackground: The aim of this study was to investigate the growth-inhibiting efficacy of Drosera intermedia extracts (water, methanol and n-hexane) against four food spoilage yeasts and five filamentous fungi strains responsible for food deterioration and associated with mycotoxin production, in order to identify potential antimycotic agents. Results: The n-hexane extract showed a broad activity spectrum against all tested microorganisms, followed, in activity, by the methanol and water extracts. The major component of the n-hexane extract was purified using a solid-phase extraction column and identified as plumbagin. Results show that high-purity plumbagin can be produced from D. intermedia cultures following a simple and effective isolation procedure. A sample of purified plumbagin was tested against the same panel of microorganisms and high growth-inhibiting capacity was observed. Minimum inhibitory concentrations less than 2 μgmL−1 were obtained against the filamentous fungi. In the case of the species Aspergillus fumigatus, A. niger and A. flavus, activities comparable to miconazole were obtained. Conclusion: The results obtained provided evidence of the antimycotic activity of plumbagin, suggesting that D. intermedia could be the source of an interesting compound for the food industry as an alternative to preservatives.
- In vitro plantlet production of the endangered Pinguicula vulgarisPublication . Grevenstuk, Tomás; Romano, AnabelaThis study describes the development of a micropropagation protocol for Pinguicula vulgaris using cultures initiated from in vitro produced seedlings. P. vulgaris is a carnivorous plant with a northern, disjunctly circumpolar distribution and specific habitat requirements, and is hence becoming increasingly rare. Shoot proliferation was significantly influenced by Murashige and Skoog (MS) macronutrient concentration, showing higher proliferation rates in 1/4MS, but was not affected by the addition of 0.1 mg/L 6-benzyladenine (BA) or zeatin (Zea). The best medium for propagating P. vulgaris was plant growth regulator (PGR) free A1/4MS. An average of 7.62 new shoots per initial explant could be obtained after 8 weeks of culture, of which over 79% produced roots during proliferation. Moreover, rooting percentages of 100% were obtained for the initial explants in all the tested media, including media without PGRs. The plantlets were successfully acclimatized to ex vitro conditions, exhibiting normal development.
- Identification of the tri-Al tricitrate complex in Plantago almogravensis by hydrophilic interaction LC with parallel ICP-MS and electrospray Orbitrap MS/MS detectionPublication . Grevenstuk, Tomás; Flis, Paulina; Ouerdane, Laurent; Lobinski, Ryszard; Romano, AnabelaThe identification of the ligands binding Al is essential to understand the mechanisms by which plants detoxify Al internally. However, studies concerning the speciation of Al have been frustrated by its complex chemistry. This work describes the identification of the tri-Al tricitrate (Al(3)cit(3)) complex in Plantago almogravensis, encompassing an integrated mass spectrometry approach based on hydrophilic interaction liquid chromatography (HILIC) and parallel detection by ICP-MS and ESI-MS/MS. This work also reports that both Al and Fe are bound by tricitrate, sometimes simultaneously, and the consequences of this finding are discussed. Of the complexes separated by size exclusion chromatography, Al(3)cit(3) is the most stable occurring in P. almogravensis as it was the only one recovered after HILIC. This approach provided new information on the mechanism of Al detoxification in P. almogravensis, namely that Al is bound by the organic acid citrate and that the relative concentration of the detected complexes is affected by the organ type and internal Al concentration, and has potential for studying the speciation of Al in less tolerant plants.
- Antioxidant activity and verbascoside content in extracts from two uninvestigated endemic Plantago spp.Publication . Gonçalves, Sandra; Grevenstuk, Tomás; Martins, Neusa; Romano, AnabelaThis work describes for the first time the antioxidant activity of extracts from wild plants and in vitro cultures of two previously uninvestigated Plantago spp. endemic to Portugal, Plantago algarbiensis Samp. and Plantago almogravensis Franco. The Well-known and widely distributed species Plantago lagopus L. was also studied for comparison. Although the extracts from the three species exhibited radical scavenging and iron-reducing properties, the extracts from the two endemic species showed greater activity irrespectively of plant material source. Both P. algarbiensis extracts showed similar antioxidant activity whereas, in the case of P. almogravensis higher activity was observed for extracts from wild plants compared to extracts from in vitro cultures. Liquid chromatography-diode array detection analyses indicate verbascoside as a major constituent of all extracts and the highest content was obtained from in vitro cultures algarbiensis. In addition, a high correlation-between-the anti-oxidant-activity measured with three. methods and the total phenolic content was found, but no correlation was observed between antioxidant activity and verbascoside content. The results suggest that P. algarbiensis and P. almogravensis are sources of health-beneficial phytochemicals and indicate that in vitro cultures represent a promising alternative for the biosynthesis of verbascoside. (C) 2014 Elsevier B.V. All rights reserved.
- In vitro propagation of Drosera intermedia in a single stepPublication . Grevenstuk, Tomás; Coelho, Natacha; Gonçalves, Sandra; Romano, AnabelaA simple and efficient protocol for the micropropagation of Drosera intermedia, using cultures initiated from in vitro produced seedlings, is described. Shoot proliferation was significantly influenced by Murashige and Skoog (MS) macronutrient concentration, showing higher multiplication rates for ¼ MS (the lowest concentration), but was not affected by the addition of 0.1 mg dm-3 kinetin. In all cases a multiplication percentage above 90 % was recorded. High rooting percentages (up to 100 %) were obtained in multiplication phase on ¼ MS medium without growth regulators. In average 15.8 plantlets per initial shoot was produced after 8 weeks of culture. All plantlets were successfully acclimatized to ex vitro conditions, exhibiting normal development.