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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.
- Field performance of seedlings and microproppagated plants of carob treePublication . Osório, Maria Leonor; Osório, Júlio; David, Maria Manuela; Romano, AnabelaMicropropagated plantlets of cv. Mulata and cv. Galhosa growing in pots, after acclimatization in the glasshouse and growth for several months under natural conditions, were transferred to a field trial. Carob seedlings of ‘Mulata’ were also transferred under the same conditions. The field trial has been established with 100% of success in micropropagated plants and 97% in seedlings. Three months after transfer to the field, plants showed good growth but micropropagated plants of ‘Mulata’ exhibited more vigour than ‘Galhosa’ and than seedlings, with the greatest number of branches and a larger length increase in the main stem. Before transference to the field, net photosynthetic rates (PN), water use efficiency (PN/gs) and quantum yield of PSII (fPSII) of the potted plants were higher in seedlings than in micropropagated plants. Three months after transplantation all gas exchange parameters were improved for all types of plants, particularly net photosynthetic rate, and no significant differences were observed between plants either micropropagated or seedlings. On the other hand, fPSII decreased significantly in seedlings. The results suggest that the pre-acclimation, in pots, to external environmental conditions might have improved field survival, plant vigour and enhanced the endurance to adverse conditions of micropropagated carob trees.
- 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.
- Antioxidant activity and in vitro inhibition of tumor cell growth by leaf extracts from the carob tree (Ceratonia siliqua)Publication . Custódio, Luísa; Fernandes, Eliana; Escapa, Ana Luísa; López-Avilés, Sandra; Fajardo, Alba; Aligué, Rosa; Alberício, Fernando; Romano, AnabelaThe methanol leaf extracts of female cultivars of the carob tree [Ceratonia siliqua L. (Fabaceae)] and of hermaphrodite and male trees were investigated for their contents of phenolic compounds, their in vitro antioxidant activity, measured by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging and linoleic acid system assays, and their in vitro tumor growth inhibition on HeLa cells. The different cultivars and trees showed high levels of phenols, and considerable variations in the amount of these compounds. The extracts showed significant radical scavenging activity (RSA), which was not significantly affected by the gender of the tree. From the female cultivars tested, Galhosa exhibited the highest RSA. Gender significantly affected the antioxidant activity of the extracts measured by the linoleic acid system assay, and males and hermaphrodites showed the highest activities. The extracts displayed a remarkable ability to inhibit tumor cell proliferation, and their bioactivity varied with different cultivars or trees tested. Extracts from male and hermaphrodite trees exhibited higher capacity to inhibit the proliferation of HeLa cells than the female cultivars.
- In vitro spore germination of Polystichum drepanum, a threatened fern from Madeira IslandPublication . Chá-Chá, R.; Fernandes, F.; Romano, AnabelaPolystichum drepanum (Sw) C. Presl is a threatened fern endemic to a few forest areas in the north-west of Madeira Island. The aims of this work were to establish suitable culture conditions for in vitro germination of spores, and to evaluate short-term storage conditions for P drepanum spores. The highest frequency of germination was obtained in Murishage and Skoog (MS) liquid medium, without agitation. However, gametophytes maintained in MS liquid medium did not grow and, after 4 weeks, became anoxic and died. Thus, after germination in liquid medium, gametophytes were transferred to an MS double-phase culture system for further growth. The effects of storage period, temperature, and relative humidity during storage on in vitro spore germination were studied. Spore viability was assessed after 2, 4 and 6 months, and high viability (> 94%) was observed in all the assays. However, germination capability decreased with increased storage periods. The number of sporophytes obtained also decreased with prolonged storage periods. The results indicate that spores of R drepanum stored for 4 months at 21 degrees C maintain high viability and high germination frequency. ne sporophytes obtained were acclimatised in a mixture of peat and vermiculite [2:1 (v/v)] under high relative humidity (90-95%). Seventy-five sporophytes were successfully acclimatised to ex vitro conditions and showed active growth in the glasshouse.
- Lignin extraction from waste pine sawdust using a biomass derived binary solvent systemPublication . Magalhães, Solange; Filipe, Alexandra; Melro, Elodie; Fernandes, Catarina; Vitorino, Carla; Alves, Luís; Romano, Anabela; Rasteiro, Maria G.; Medronho, Bruno; MDPILignocellulosic biomass fractionation is typically performed using methods that are somehow harsh to the environment, such as in the case of kraft pulping. In recent years, the development of new sustainable and environmentally friendly alternatives has grown significantly. Among the developed systems, bio-based solvents emerge as promising alternatives for biomass processing. Therefore, in the present work, the bio-based and renewable chemicals, levulinic acid (LA) and formic acid (FA), were combined to fractionate lignocellulosic waste (i.e., maritime pine sawdust) and isolate lignin. Different parameters, such as LA:FA ratio, temperature, and extraction time, were optimized to boost the yield and purity of extracted lignin. The LA:FA ratio was found to be crucial regarding the superior lignin extraction from the waste biomass. Moreover, the increase in temperature and extraction time enhances the amount of extracted residue but compromises the lignin purity and reduces its molecular weight. The electron microscopy images revealed that biomass samples suffer significant structural and morphological changes, which further suggests the suitability of the newly developed bio-fractionation process. The same was concluded by the FTIR analysis, in which no remaining lignin was detected in the cellulose-rich fraction. Overall, the novel combination of bio-sourced FA and LA has shown to be a very promising system for lignin extraction with high purity from biomass waste, thus contributing to extend the opportunities of lignin manipulation and valorization into novel added-value biomaterials.
- 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.