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- 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.
- 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.
- 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.