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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.
- 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.
- 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.
- 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.
- 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.