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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.
- 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.
- 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.
- 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.
- Effects of CO2 concentration on acclimatization and physiological responses of two cultivars of carob treePublication . Osório, Maria Leonor; Gonçalves, Sandra; Osório, Júlio; Romano, AnabelaThis study reports survival and physiological responses of micropropagated Ceratonia siliqua L. cvs. Galhosa and Mulata plants during ex vitro acclimatization under ambient (AC; 330 μmol mol-1) or elevated (EC; 810 μmol mol-1) CO2 concentration and a photosynthetic photon flux density of 125 μmol m-2 s-1. CO2 enrichment during acclimatization did not improve survival rate that was around 80 % for both treatments. Eight weeks after ex vitro transplantation, photosynthetic capacity and apparent quantum yield in acclimatized leaves were higher in comparison with those in in vitro-grown leaves, without any significant difference between CO2 treatments. Chlorophyll content increased after acclimatization. However, EC led to a decrease in the total amount of chlorophyll in new leaves of both cultivars, compared to those grown at AC. Soluble sugars and starch contents were not markedly affected by growth EC, although starch had significantly increased after transfer to ex vitro conditions. EC induced an increase in the stem elongation and in the effective life of leaves, and a decrease in the number of new leaves.
- Establishment of an in vitro propagation protocol for Thymus lotocephalus, a rare aromatic species of the Algarve (Portugal)Publication . Coelho, Natacha; Gonçalves, Sandra; González-Benito, María Elena; Romano, AnabelaThe aim of this work was to develop an in vitro propagation protocol for the endangered species Thymus lotocephalus using seedlings as explants. Several macronutrient concentrations of Murashige and Skoog medium (MS), cytokinin types and concentrations, and cytokinin/auxin combinations were tested to assess the shoots’ proliferation capacity. Although the best proliferation results were obtained with 6-benzyladenine, high percentages of hyperhidric shoots were observed. Because high proliferation of healthy shoots was observed in MS medium that was free of plant growth regulators, this medium was chosen for proliferation studies. The best rooting results were achieved in MS medium without auxins (92.00 ± 6.11%, 6.54 ± 0.52 and 1.60 ± 0.10 cm regarding rooting frequency, number of roots per shoot and longest roots, respectively) or supplemented with 0.5 mg l-1 indole-3-acetic acid (98.00 ± 2.11%, 11.14 ± 0.75 and 2.40 ± 0.24 cm, respectively). Plantlets were successfully acclimatised to ex vitro conditions with a survival rate of 93.33%. With the development of this micropropagation protocol, an important contribution has been made to the conservation of the endangered species T. lotocephalus.
- Plantas silvestres comestíveis do AlgarvePublication . Romano, Anabela; Gonçalves, SandraA região do Algarve, em virtude da sua situação geográfica privilegiada e condições edafo-climáticas particulares, é um dos centros de maior diversidade botânica do país. Para além de um número considerável de espécies nativas existem, no elenco florístico da região, diversas espécies de diferentes proveniências, mais ou menos longínquas, que se encontram naturalizadas (sem comportamento invasor) e algumas em estado assilvestrado. Este conjunto de espécies espontâneas, que compõem e caracterizam a paisagem algarvia, constitui um valor patrimonial a preservar. A conservação dos recursos genéticos é uma preocupação comum da humanidade e parte integrante do processo de desenvolvimento económico e social. Na atualidade esta é uma das questões mais pertinentes e controversas que a humanidade enfrenta. Em plena mudança global, onde os ecossistemas enfrentam grandes mudanças climáticas e fortes pressões antropogénicas, a erosão e perda de diversidade genética é uma realidade cada vez mais notória. A conservação dos recursos genéticos é, assim, uma prioridade estabelecida e reconhecida a nível mundial. O reconhecimento da necessidade de uma ação internacional concertada para fazer frente à perda e redução da biodiversidade, levou à criação da Convenção sobre a Diversidade Biológica (CDB) assumindo três objetivos fundamentais: a conservação da diversidade biológica; a utilização sustentável dos seus componentes; e a partilha justa e equitativa dos benefícios provenientes da utilização dos recursos genéticos. Em harmonia com a CDB também o Tratado Internacional sobre os Recursos Fitogenéticos para a Alimentação e a Agricultura, da Organização das Nações Unidas para a Alimentação e a Agricultura (FAO), veio reconhecer a importância da conservação e uso sustentável dos recursos genéticos vegetais para a alimentação, a agricultura e a partilha justa e equitativa derivada do seu uso, para uma agricultura sustentável e segurança alimentar.