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- Chemical profiling and biological screening of Thymus lotocephalus extracts obtained by supercritical fluid extraction and hydrodistillationPublication . Costa, Patrícia; Gonçalves, Sandra; Grosso, Clara; Andrade, Paula B.; Valentão, Patrícia; Bernardo-Gil, Maria Gabriela; Romano, AnabelaEssential oil and extracts from the aerial parts of Thymus lotocephalus were obtained by hydrodistillation (HD) and supercritical fluid extraction (SFE) in two different collectors, respectively. SFE was conducted at 40 ◦C and a working pressure of 12 or 18 MPa. The chemical profiles were determined using GC-FID and GC–IT–MS. Oxygen-containing monoterpenes were the primary constituents in the essential oil and SFE extracts collected in the second separator, while the extracts obtained in the first separator were predominantly oxygen-containing sesquiterpenes. A large number of compounds were identified by hydrodistillation and, in contrast, the highest extraction yields were obtained using SFE. Linalool(10.43 ± 1.63%) was the main component in essential oil, whereas camphor (7.91 ± 0.84%) and cis-linalool oxide (7.25 ± 1.45%) were the major compounds in the extracts-2nd separator obtained at pressures of 12 and 18 MPa, respectively. Caryophyllene oxide was the primary constituent identified in the extracts-1st separator (4.34 ± 0.51 and 4.41 ± 1.25% obtained at 12 and 18 MPa, respectively). The antioxidant activity was assessed by ORAC and DPPH assays, and the anti-cholinesterase activity was evaluated in vitro using Ellman’s method. The essential oil and SFE extracts (first separator) of T. lotocephalus possessed antioxidant activity and strongly inhibited cholinesterases. We also demonstrated that the acetylcholinesterase and butyrylcholinesterase inhibitory activities of the essential oil could be attributed to 1,8-cineole and caryophyllene oxide, respectively.
- Genetic diversity of wild populations of Tuberaria major (Cistaceae), an endangered species endemic to the Algarve region (Portugal), using ISSR markersPublication . Trindade, Helena; Sena, Inês; Gonçalves, Sandra; Romano, AnabelaTuberaria major (Willk.) P. Silva & Rozeira (Cistaceae) is an endangered species endemic to the Algarve region (Portugal). Due to the increasing urban pressure, natural populations are now reduced to small nuclei located in the center of the region. Genetic variation within and among three populations of this species was evaluated using inter-simple sequence repeat (ISSR) markers. A total of eight primers generated 128 well defined bands, with an average of 16 bands per primer. At the population level, the percentage of polymorphic bands ranged from 55 to 73%, while at the species level this value increased to 98%. The population showing the highest genetic variability was Olhão and the lowest was Gambelas. According to the cluster analysis based on UPGMA, the population Gambelas was clearly separated from the other two, which clustered together. The three populations revealed a high level of genetic diversity, with a Nei’s genetic diversity of 0.197 and Shannon’s diversity index of 0.324. A high level of gene flow was observed among populations (Nm ¼ 2.199), reflecting a moderate level of genetic differentiation (GST ¼ 0.185), and no correlation between genetic and geographical distances (r ¼ 1.000;P > 0.05). Based on the results obtained it is concluded that the Gambelas population should be a priority for conservation, including the ex situ propagation and restoration.
- 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.
- Physiological traits and oxidative stress markers during acclimatization of micropropagated plants from two endangered Plantago species: P-algarbiensis Samp. and P-almogravensis FrancoPublication . Gonçalves, Sandra; Martins, Neusa; Romano, A.Plantago algarbiensis Samp. and Plantago almogravensis Franco are species endemic to Portugal at risk of global extinction. The aim of this study was to investigate the ex vitro performance of micropropagated P. algarbiensis and P. almogravensis plants in terms of survival, relative water content (RWC), photosynthetic pigment contents, H2O2 accumulation, activities of superoxide dismutase (SOD) and catalase (CAT), lipid peroxidation, and soluble protein content, in comparison with wild-grown plants. Relatively high survival rates and RWC values during the acclimatization process were observed for both species. In P. algarbiensis, the pigment content increased when plantlets were transferred to ex vitro conditions, indicating enhanced light absorption capacity. No significant alterations in H2O2 content, CAT activity, or lipid peroxidation level were observed during acclimatization, but the protein content decreased in plants at the end of the growth chamber and greenhouse stages. When P. almogravensis plantlets were transferred to the ex vitro environment, decreases in the H2O2 content were observed that correlated to increased CAT activity and SOD maintenance, which lead to decreased lipid peroxidation and protein content. It was concluded that micropropagated P. algarbiensis and P. almogravensis plants were able to manage the oxidative stress induced by the in vitro environment and to perform well under ex vitro conditions.
- Galangal, the multipotent super spices: A comprehensive reviewPublication . Das, Gitishree; Patra, Jayanta Kumar; Gonçalves, Sandra; Romano, Anabela; Gutierrez-Grijalva, Erick P.; Heredia, J. Basilio; Das Talukdar, Anupam; Shome, Soumitra; Shin, Han-SeungBackground: Galangal is highly appreciated for its potential applications in food and medicine. In spite of its usage in food and herbal medicine in countries like Indonesia and Thailand, very limited data is accessible about its potential applications in therapeutics and pharmacology. It has been reported to be utilized in herbal medicines for treatment against diseases like hemorrhoids, abnormal menstruation, abdominal discomfort, and inflammation, among others. Scope and approach: The widespread use of galangal in food and traditional medicine has fascinated researchers all over the world looking for its enhanced medicinal capabilities, new bioactive compounds with nutraceutical potentials, and probable uses in medicine and pharmacology. A number of literature search methods like searching the related contents with the keywords such as Alpinia sp., galangal, antiviral, cardiovascular, neuroprotective, bioactive compounds, phytochemicals etc. in the web search engines' such as the Google search, Web of Science, Scopus, PubMed etc. were implemented. Key findings and conclusions: Conversely, there is no recent structured and ample review that summarizes the available data on these issues. In the current review, these topics are addressed in detail along with the investigation of scientific evidence supporting the vast applications of galangal in food and its medicinal properties, such as antiviral, cardiovascular and neuroprotective properties, together with the preclinical and clinical studies with galangal bioactive compounds.
- Effect of in vitro gastrointestinal digestion on the total phenolic contents and antioxidant activity of wild Mediterranean edible plant extractsPublication . Gonçalves, Sandra; Moreira, E.; Andrade, Paula B.; Valentao, Patricia; Romano, AnabelaThe recent interest in wild edible plants is associated with their health benefits, which are mainly due to their richness in antioxidant compounds, particularly phenolics. Nevertheless, some of these compounds are metabolized after ingestion, being transformed into metabolites frequently with lower antioxidant activity. The aim of the present study was to evaluate the influence of the digestive process on the total phenolic contents and antioxidant activity of extracts from four wild edible plants used in the Mediterranean diet (Beta maritima L., Plantago major L., Oxalis pes-caprae L. and Scolymus hispanicus L.). HPLC-DAD analysis revealed that S. hispanicus is characterized by the presence of caffeoylquinic acids, dicaffeoylquinic acids and flavonol derivatives, P. major by high amounts of verbascoside, B. maritima possesses 2,4-dihydroxybenzoic acid, 5-O-caffeoylquinic acid, quercetin derivatives and kaempferol-3-O-rutinoside, and O. pes-caprae extract contains hydroxycinnamic acids and flavone derivatives. Total phenolic contents were determined by Folin-Ciocalteu assay, and antioxidant activity by the ABTS, DPPH, ORAC and FRAP assays. Phenolic contents of P. major and S. hispanicus extracts were not affected by digestion, but they significantly decreased in B. maritima after both phases of digestion process and in O. pes-caprae after the gastric phase. The antioxidant activity results varied with the extract and the method used to evaluate the activity. Results showed that P. major extract has the highest total phenolic contents and antioxidant activity, with considerable values even after digestion, reinforcing the health benefits of this species.
- Morphological, physiological and oxidative stress markers during acclimatization and field transfer of micropropagated Tuberaria major plantsPublication . Osório, Maria Leonor; Gonçalves, Sandra; Coelho, N.; Osório, Júlio; Romano, AnabelaTuberaria major (Willk.) P. Silva and Rozeira is a critically-endangered rock rose species endemic to Portugal. Because the species needs to be preserved, this study evaluated the morphological and physiological traits of micropropagated T. major plants during acclimatization and field transfer. There were no significant differences between wild and micropropagated plants in the field, although the latter underwent significant changes during acclimatization. Leaf pubescence and leaf mass per area increased during acclimatization whereas the chlorophyll content and chlorophyll/carotenoid ratio declined to eventually match those of wild plants. Stomatal conductance (g(s)) and transpiration rates (E) also declined substantially during acclimatization, thus preventing uncontrolled wilting. Photosynthetic rate (P-N) was initially negative but increased during the later stages of acclimatization. Maximum quantum yield of PSII (F-v/F-m) remained constant at 0.78-0.85, showing that the plants were healthy and unstressed. PSII quantum efficiency (I center dot(PSII)) was initially low but increased during acclimatization along with photosynthetic performance as the energy partitioning in PSII was adjusted. This was balanced by the decline in non-regulated energy dissipation (I center dot(NO)) from an initially high value. Electrolyte leakage and malondialdehyde content remained constant at similar levels in both groups of plants, but H2O2 levels were higher in the field, perhaps indicating the early induction of antioxidant defense systems. The present study shows that T. major has enough phenotypic plasticity to adapt to changing environments and that the procedure described herein can be used for the restoration and preservation of this species.
- Extraction of antioxidants from winemaking byproducts: effect of the solvent on phenolic composition, antioxidant and anti-cholinesterase activities, and electrochemical behaviourPublication . Jara-Palacios, María José; Gonçalves, Sandra; Heredia, Francisco J.; Hernanz, Dolores; Romano, AnabelaExtraction solvent is a very important factor in the recovery of antioxidants from natural matrices. In this study, the effect of three solvents (ethanol, ethanol/water and water) on the phenolic composition, antioxidant and anti-cholinesterase activities and electrochemical behaviour of four winemaking byproducts (seeds, skins, stems, and pomace) was evaluated. Phenolic composition was determined by the Folin-Ciocalteu method and ultra-high-performance liquid chromatography (UHPLC), antioxidant activity by the capacity to scavenge 2,2-diphenyl-1-picrylhydrazyl and hydroxyl radicals, anti-cholinesterase activity by the Ellman's method, and electrochemical behaviour by cyclic voltammetry. Eight phenolic compounds were quantified with higher content in water/ethanol extracts (e.g., epicatechin in pomace: 17 mg/100 g vs. 7 and 6 mg/100 g in ethanol and water extracts, respectively), although there were some exceptions (e.g., gallic acid in seeds was most abundant in water extracts). Moreover, the highest total phenolic content (TPC) and antioxidant activity were found in ethanol/water extracts (between 2 and 30-fold the values of the other extracts). Overall, the most active extracts in inhibiting both acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes were ethanol/water and ethanol extracts from seeds (between 31.11 and 53.90%). The electrochemical behaviour allowed for differentiating the extracts depending on the solvent and the byproduct. Our findings indicate that winemaking byproducts represent a source of phenolic compounds with antioxidant and anti-cholinesterase activities and suggest that cyclic voltammetry is a promising technique to evaluate the phenolic extraction process from these byproducts.
- Phenolic profile, antioxidant activity and enzyme inhibitory activities of extracts from aromatic plants used in Mediterranean dietPublication . Goncalves, Sandra; Moreira, Elsa; Grosso, C.; Andrade, Paula B.; Valentao, Patricia; Romano, AnabelaThe antioxidant and enzyme inhibitory properties of methanolic extracts from four aromatic plants used in traditional medicine and food [Calamintha nepeta (L.) Savi subsp. nepeta, Helichrysum italicum subsp. picardii Franco, Mentha spicata L. and Origanum vulgare subsp. virens (Hoffmanns. & Link) Bonnier & Layens] were evaluated. The extract from O. vulgare exhibited the strongest DPPH (IC50 of 4.65 +/- 0.12 mu g/ml) and ABTS (1479.56 +/- 12.29 mu mol(TE)/g(extract)) scavenging capacities, as well as the largest ferric reducing ability (1746.76 +/- 45.11 mu mol(AAE)/g(extract)). This extract also showed the highest total phenolic content (1597.20 +/- 24.10 mu mol(GAE)/g(extract)) and although HPLC-DAD analysis revealed rosmarinic acid as the main compound of the extract, other compounds seem to be involved in the antioxidant activity. Furthermore, the extract from H. italicum, which was found to be rich in caffeoylquinic and dicaffeoylquinic acids and in pinocembrin, showed the highest inhibitory potential against acetylcholinesterase, tyrosinase and alpha-glucosidase. Overall, the results obtained validate the usefulness of the studied plants as valuable sources of natural agents beneficial for human health.
- High-frequency in vitro propagation of the endangered species Tuberaria majorPublication . Gonçalves, Sandra; Fernandes, Laura; Romano, AnabelaA novel protocol suitable for the micropropagation of the endangered species Tuberaria major using seedlings as explants is reported. Using this protocol, we studied the effects of explant type (apical shoots and nodal segments) and cytokinins [6-benzyladenine (BA), kinetin,and zeatin (ZEA)] on shoot proliferation. Explant type significantly influenced the proliferation frequency and mean number of shoots, with nodal segments showing a higher proliferation capacity. The mean number of shoots was significantly higher when the explants were cultured in half-strength (1/2) MS medium supplemented with 0.2 mg l-1 BA (6.83 ± 0.77 shoots) or ZEA (6.55 ± 0.71 shoots). The shoots showed a great rooting capacity that was significantly influenced by the concentration of MS macronutrients but not by the concentration of auxins. The highest rooting frequencies (97–100%) were obtained in 1/2 MS medium with or without plant growth regulators. The plants obtained were easily acclimatized to ex vitro conditions, with 97% surviving after 6 weeks. The micropropagated plants were successfully reintroduced into their natural habitat and exhibited normal development. In conclusion, our culture protocol, with efficient seed germination, subsequent multiplication of nodal explants using ZEA at 0.2 mg l-1, and successful ex vitro establishment of well-rooted plantlets on 1/2 MS medium, provides a simple and reliable methodology for the large scale propagation of T. major, thereby contributing to germplasm preservation of this endangered species.