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- Evaluation of the antioxidant and antimicrobial properties of in vitro cultured Drosera intermedia extractsPublication . Grevenstuk, Tomás; Gonçalves, Sandra; Almeida, Sara; Coelho, Natacha; Quintas, Célia; Gaspar, Maria Nelma; Romano, AnabelaEvaluation of the antioxidant activity of the methanol, water and n-hexane extracts of Drosera intermedia, determined by the Folin-Ciocalteau (F-C), trolox equivalent antioxidant capacity (TEAC) and oxygen radical antioxidant capacity (ORAC) assays showed that the methanol extract had the highest antioxidant activity (F-C: 378.6 ± 31.5 μmolGAE/mgextract; TEAC: 332.2 ± 29.1 μmolTE/mgextract; ORAC: 64.7 ± 7.8 μmolTE/mgextract). Antimicrobial activity was tested against seven bacterial and eight yeast strains using the agar diffusion assay, followed by the determination of minimum inhibitory concentrations (MIC). All tested D. intermedia extracts demonstrated strong antimicrobial properties with a broad spectrum of activity. However, the n-hexane extract exhibited much greater activity than water and methanol extracts. The most susceptible microorganisms to the n-hexane extract were Staphylococcus epidermidis ATCC 12228 and Candida albicans YP0175, for which a MIC value of 13.0 μg/mL was scored.
- 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.
- An efficient in vitro propagation protocol for Pinguicula lusitanica, a rare insectivorous plantPublication . Gonçalves, Sandra; Escapa, Ana Luísa; Grevenstuk, Tomás; Romano, AnabelaIn this study, an efficient protocol was developed for in vitro propagation of Pinguicula lusitanica L., a rare insectivorous plant with pharmacological value and limited reproductive capacity. The effects of two concentrations (0.1 and 0.5 mg l-1) of a range of plant growth regulators, including cytokinins (BA, KIN, and ZEA) and auxins (IAA, IBA, and NAA), and three concentrations of MS medium macronutrients (total, 1/2 and 1/4MS) on proliferation and rooting, were investigated. P. lusitanica shoots showed abundant proliferation and rooting capacity, both of which were significantly influenced by MS medium strength. The use of 1/2MS supplemented with 0.5 mg l-1 BA or KIN ensure a 29-fold rate of proliferation. Best rooting frequency and higher root number and length were attained in 1/4MS medium containing 0.2 mg l-1 IAA. Sixty percent of the plantlets were successfully acclimatized to ex vitro conditions, exhibiting normal development. These plantlets can be used to replenish declining populations in the wild and/or for the extraction of bioactive compounds, reducing pressure on wild stocks.
- 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.
- 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.
- Plumbagin recovery in field specimens of Drosophyllum lusitanicum (L.) LinkPublication . Grevenstuk, Tomás; Gonçalves, Sandra; Nogueira, José Manuel F.; Romano, AnabelaThe naphthoquinone plumbagin has a broad spectrum of biological activities. The aim of this study was to investigate the efficiency of two extraction methods (Soxhlet and ultrasound-assisted extraction) and three solvents (methanol, chloroform and hexane) to recover plumbagin from fresh and dried tissues of field specimens of Drosophyllum lusitanicum (L.) Link. The highest extraction yields were obtained with methanol as solvent and using fresh plant material. The obtained extracts were analysed by gas chromatography with mass spectrometric detection and plumbagin was the major compound present. Plumbagin was quantified in the extracts using the external standard methodology. The results obtained showed that the best recoveries of plumbagin were attained using fresh plant material and there were no significant differences between Soxhlet and ultrasound-assisted extraction. Moreover, hexane proved to be the more appropriate solvent for the extraction of plumbagin, providing high recoveries and the most concentrated extracts, yielding 2.42 mg of plumbagin per gram of plant material with the highest degree of purity. This method is a simple and efficient one to extract large amounts of plumbagin from D. lusitanicum field specimens.
- 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.