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- Assessment of 83 pharmaceuticals in WWTP influent and effluent samples by UHPLC-MS/MS: Hourly variationPublication . Paíga, Paula; Correia, Manuela; Fernandes, Maria João; Silva, Ana Margarida; Martins de Carvalho, Maria Manuela; Vieira, Joana; Jorge, Sandra; Silva, Jaime Gabriel; Freire, Cristina; Delerue-Matos, CristinaThe removal efficiency of pharmaceuticals in wastewater treatment plants (WWTPs) is variable and some of these compounds pass these plants almost intact and others presenting a removal efficiency close to 100%. Their incomplete removal results in a continuous discharge of pharmaceuticals into the environment. To assess the profile of contamination of influents and effluents over a day, a set of 83 pharmaceuticals were evaluated hourly in a WWTP in Leiria, Portugal. The composite samples of the influent and effluent were also collected. Concentrations varied from 1 in WWTP influents, and carbamazepine, fluoxetine, sertraline the pharmaceuticals with an RQ > 1 in WWTP effluents.
- Development and Optimization of a Topical Formulation with Castanea sativa Shells Extract Based on the Concept “Quality by Design”Publication . Oliveira, Nágilla; Cádiz-Gurrea, María de La Luz; Silva, Ana Margarida; Macedo, Catarina; Rodrigues, Francisca; Costa, PauloThe proposed study aims to develop and optimize a topical formulation with Castanea sativa shells extract considering the concept of Quality by Design, focusing on a planned development that consider the vulnerabilities of the entire process through risk analysis tools and design of experiments (DoE). A Box–Behnken design with three factors and three levels was used as a statistical tool for the execution of the DoE and the analysis of the response surface methodology responses. The independent variables studied were the quantity of sodium lauryl sulfate (%) (X1), beeswax (%) (X2) and macadamia oil (%) (X3); the dependent variables were pH (Y1), viscosity (Y2) and adhesiveness (Y3). According to the mathematical model, the optimal formulation contains 0.93% of sodium lauryl sulfate, 5.00% of beeswax and 10.00% of macadamia oil. The optimal formulation with the extract was prepared and characterized over the time, regarding organoleptic and technological characteristics, allowing conclusions to be reached regarding its stability. The formulation presented a pleasant odor and was light brown in color, it also demonstrated pseudoplastic-thixotropic behavior and a small reduction in the formulation consistency after 30 days of storage. This study demonstrated the efficiency of the Quality by Design methodology to understand the product variability, supporting that this approach favors a better understanding of the whole process and enables to design a robust development stage, reducing costs and generating high-quality products
- Eco-friendly insights on kiwiberry leaves valorization through in-vitro and in-vivo studiesPublication . Silva, Ana Margarida; Garcia, Juliana; Dall'Acqua, Stefano; Costa, Paulo; Delerue-Matos, Cristina; Rodrigues, FranciscaOn the last decade, the demand for novel active skin ingredients based on sustainable principles arise as a trend. These new concerns transformed the consumer preferences, which are not actually solved by the industry portfolio. Regulatory aspects establish guidelines to assess the safety and efficacy of new cosmetic ingredients, being this step imperative to introduce a new ingredient on the market. Our last studies reported the richness of Actinidia arguta (kiwiberry) leaves in bioactive compounds with potential skin applications, but further studies are necessary to validate this extract. Therefore, the current study aimed to screen the hydroalcoholic extract of A. arguta leaves regarding elastase and hyaluronidase inhibition, achieving excellent results (65.62 ± 2.09% and 54.64 ± 4.17%, respectively). The cell viability was assessed in keratinocytes and fibroblasts (0.1–1000 μg/mL), ensuring the absence of cytotoxic effects. Afterwards, 3D models, namely a skin (EpiSkin™) and an ocular (SkinEthic™ HCE, respectively) model, were used to estimate the irritant potential. The viability achieved in both models after exposure to the extract was, respectively, 55.18 ± 10.60% and 101.15 ± 5.96%. The IL-1α released for the skin and ocular models were, respectively, 0 pg/mL and 35.60 ± 2.61 pg/mL, being the extract classified as non-irritant for both models. Lastly, in-vivo studies performed through a patch test in 10 human volunteers proved the absence of allergic or irritative reactions. Overall, the results attained highlighted that the hydroalcoholic kiwiberry leaves extract is an excellent cosmetic ingredient, being not irritant to skin application
- Assessment of a Formulation Containing a Castanea sativa Shells Extract on Skin Face Parameters: In Vivo EvaluationPublication . Silva, Ana Margarida; Costa, Paulo C.; Delerue-Matos, Cristina; Rodrigues, FranciscaIn the last years, the demand for new eco-friendly ingredients has increased on the cosmetic market. Consumers are more aware of sustainable principles and, simultaneously, more conscious regarding the skin aging process. Chestnut (Castanea sativa) shells are a food by-product produced in high quantities in Europe. This waste has been described as a rich source of phenolic compounds with skin biological effects, such as antioxidant, antiradical, and anti-inflammatory activities. Despite the huge number of assays reporting the richness of chestnut shell extracts in bioactive compounds as well as the development of cosmetic formulations containing these extracts, no in vivo assays have assessed their clinical efficacy in human volunteers. The aim of this study was to evaluate the effect on skin face parameters of a formulation containing a chestnut shell extract in human volunteers (n = 22) who applied the product twice per day, for 56 days. For that, biophysical techniques, including Corneometer®, Cutometer®, and PrimosPremium, were employed, allowing the quantification of skin hydration and firmness, as well as of wrinkles’ depth and volume and wrinkles’ skin roughness. The results demonstrated that the formulation led to a slight decrease in roughness and wrinkles’ depth, although no significant differences with respect to a placebo were observed. In addition, a clear improvement of skin hydration was achieved (t0 = 54.00 Arbitrary Units (A.U.) and t56 = 58.62 A.U.). In contrast to the placebo, the active formulation increased skin firmness up to 31.76% in 50.00% of the volunteers, but without significant differences with respect to the placebo, probably due to the short period of treatment. A long-term use of the product is recommended to possibly observe significant differences in all parameters.
- Valorization of Kiwiberry Leaves Recovered by Ultrasound-Assisted Extraction for Skin Application: A Response Surface Methodology ApproachPublication . Silva, Ana Margarida; Pinto, Diana; Moreira, Manuela M.; Costa, Paulo C.; Delerue-Matos, Cristina; Rodrigues, FranciscaThis study aims to evaluate the optimal ultrasound-assisted extraction (UAE) conditions of antioxidants polyphenols from Actinidia arguta (Siebold & Zucc.) Planch. Ex Miq. (kiwiberry) leaves using a response surface methodology (RSM). The effects of solid:liquid ratio (2.5–10.0% w/v), time (20–60 min), and intensity (30–70 W/m2) on the total phenolic content (TPC) and antioxidant/antiradical activities were investigated. The optimal UAE conditions were achieved using a solid:liquid ratio of 10% (w/v) and an ultrasonic intensity of 30 W/m2 for 31.11 min. The results demonstrated that the optimal extract showed a high TPC (97.50 mg of gallic acid equivalents (GAE)/g dw) and antioxidant/antiradical activity (IC50 = 249.46 µg/mL for ABTS assay; IC50 = 547.34 µg/mL for DPPH assay; 1440.13 µmol of ferrous sulfate equivalents (FSE)/g dw for ferric reducing antioxidant power (FRAP)) as well as a good capacity to scavenge superoxide and hypochlorous acid (respectively, IC50 = 220.13 μg/mL and IC50 =10.26 μg/mL), which may be related with the 28 phenolic compounds quantified. The in vitro cell assay demonstrated that the optimal extract did not decrease the keratinocytes’ (HaCaT) viability, while the fibroblasts’ (HFF-1) viability was greater than 70.63% (1000 µg/mL). This study emphasizes the great potential of kiwiberry leaves extracted by UAE for skin application.
- New Perspectives on the Sustainable Employment of Chestnut Shells as Active Ingredient against Oral Mucositis: A First ScreeningPublication . Ferreira, Ana Sofia; Silva, Ana Margarida; Pinto, Diana; Moreira, Manuela M.; Ferraz, Ricardo; Švarc-Gajić, Jaroslava; Costa, Paulo C.; Delerue-Matos, Cristina; Rodrigues, FranciscaOral mucositis (OM), a common side effect of oncological treatment, is an oral mucosal disorder characterized by painful ulcerations and increased risk of infection. The use of natural antioxidants to suppress the redox imbalance responsible for the OM condition has emerged as an interesting approach to prevent/treat OM. This study aims to explore the chestnut (Castana sativa) shells as potential active ingredient against OM. Therefore, chestnut shells were extracted at different temperatures (110–180 °C) by Subcritical Water Extraction (SWE), aiming to recover antioxidants. The extracts were also evaluated against microorganisms present in the oral cavity as well as on human oral cell lines (TR146 and HSC3). The highest phenolic content was obtained with the extraction temperature of 110 °C, exhibiting the best antioxidant/antiradical activities and scavenging efficiencies against HOCl (IC50 = 4.47 μg/mL) and ROO• (0.73 μmol TE/mg DW). High concentrations of phenolic acids (e.g., gallic and protocatechuic acids) and flavanoids (catechin, epicatechin and rutin) characterized the phenolic profile. The antimicrobial activity against several oral microorganisms present in the oral cavity during OM, such as Streptococcus, Staphylococcus, Enterococcus, and Escherichia, was demonstrated. Finally, the effects on HSC3 and TR146 cell lines revealed that the extract prepared at 110 °C had the lowest IC50 (1325.03 and 468.15 µg/mL, respectively). This study highlights the potential effects of chestnut shells on OM
- Chemical composition and bioactivity of oilseed cake extracts obtained by subcritical and modified subcritical waterPublication . Švarc-Gajić, Jaroslava; Rodrigues, Francisca; Moreira, Manuela M.; Delerue-Matos, Cristina; Morais, Simone; Dorosh, Olena; Silva, Ana Margarida; Bassani, Andrea; Dzedik, Valentin; Spigno, GiorgiaRecovery of bioactive compounds from biowaste is gaining more and more interest in circular economy models. The oilseed cakes are usually insufficiently exploited by most technologies since they represent valuable matrices abundant in proteins, minerals, and phytochemicals, but their use is mostly limited to feed ingredients, fertilizers or biofuel production. This study was thus focused on the exploration of new valorization pathways of oilseed cakes by subcritical water, representing a safe and economic alternative in the creation of value chains. Pumpkin, hemp, and flax seed cakes were treated with subcritical water in nitrogen and carbon-dioxide atmospheres, as well as in nitrogen atmosphere with the addition of acid catalyst. The degradation of carbohydrate fraction was studied by quantifying sugars and sugar degradation products in the obtained extracts. The extracts obtained under different conditions were further compared chemically with respect to total phenols and flavonoids, as well as to the content of individual phenolic compounds. Furthermore, the effects of subcritical water treatment conditions on antioxidant, antiradical and cytotoxic properties of thus obtained extracts were defined and discussed.
- Insights into the 3D In Vitro Permeability and In Vivo Antioxidant Protective Effects of Kiwiberry Leaf Extract: A Step Forward to Human Nutraceutical UsePublication . Silva, Ana Margarida; Almeida, Andreia; Dall’Acqua, Stefano; Loschi, Francesca; Sarmento, Bruno; Costa, Paulo C.; Delerue-Matos, Cristina; Rodrigues, FranciscaActinidia arguta (Siebold & Zucc.) Planch. ex Miq. (kiwiberry) leaves are a source of phenolic compounds with pro-health biological effects, such as antioxidant and anti-inflammatory activities. Despite the huge number of studies reporting the composition of A. arguta leaves, no in vitro or in vivo studies explore its potential use as nutraceutical ingredient based on these activities. Therefore, this study aims to characterize the safety profile of kiwiberry leaf extracts using in vitro and in vivo approaches through the assessment of intestinal cell viability (Caco-2 and HT29-MTX), 3D intestinal permeation, and, most important, the redox markers, biochemical profile and liver and kidney function effects after the animal assays. Briefly, wistar rats were orally treated for 7 days with kiwiberry leaf extracts (50 and 75 mg/kg bw), water (negative control), or vitamin C (positive control). The cell viability was above 90% at 1000 μg/mL for both cells. Coumaroyl quinic acid and rutin achieved a permeation higher than 25% in the 3D intestinal model. The animal studies confirmed the extracts’ ability to increase superoxide dismutase, glutathione peroxidase, and catalase content in animals’ livers and kidneys while simultaneously decreasing the triglycerides content. This study highlighted the antioxidant capacity of kiwiberry leaf extracts, ensuring their efficacy and safety as a nutraceutical ingredient
- Salicornia ramosissima: A New Green Cosmetic Ingredient with Promising Skin EffectsPublication . Correia, Ana; Silva, Ana Margarida; Moreira, Manuela M.; Salazar, Miguel; Švarc-Gajić, Jaroslava; Brezo-Borjan, Tanja; Cádiz-Gurrea, Maria de la Luz; Carretero, Antonio Segura; Loschi, Francesca; Dall’Acqua, Stefano; Delerue-Matos, Cristina; Rodrigues, FranciscaThis study aims to validate a new cosmetic ingredient from Salicornia ramosissima S J. Woods through in vitro and ex vivo assays. The halophyte extracts were obtained by subcritical water extraction (SWE) at different temperatures (110, 120, 140, 160 and 180 °C). The antioxidant/radical scavenging activities and the phenolic profile were screened for all extracts. The optimal extract was assessed in keratinocytes and fibroblasts, while permeation assays were performed in Franz cells. The inhibitory activity of hyaluronidase and elastase was also evaluated. The sample extracted at 180 °C presented the highest phenolic content (1739.28 mg/100 g of dry weight (dw)). Despite not being efficient in the sequestration of ABTS•+, this extract scavenged the DPPH• (IC50 = 824.57 µg/mL). The scavenging capacity of superoxide (O2•−) and hypochlorous acid (HOCl) was also considerable (respectively, IC50 = 158.87 µg/mL and IC50 = 5.80 µg/mL). The cell viability assays confirmed the absence of negative effects on keratinocytes, while the fibroblasts’ viability slightly decreased. The ex vivo permeation of rutin, quercetin and syringic acid after 24 h was, respectively, 11, 20 and 11%. Additionally, the extract showed a good elastase and hyaluronidase inhibitory activity. The results obtained support the S. ramosissima bioactivity as a cosmetic ingredient.
- Formulation, Characterization, and Cytotoxicity Evaluation of Lactoferrin Functionalized Lipid Nanoparticles for Riluzole Delivery to the BrainPublication . Teixeira, Maria Inês; Lopes, Carla Martins; Gonçalves, Hugo; Catita, José; Silva, Ana Margarida; Rodrigues, Francisca; Amaral, Maria Helena; Costa, Paulo C.Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with a very poor prognosis. Its treatment is hindered by a lack of new therapeutic alternatives and the existence of the blood–brain barrier (BBB), which restricts the access of drugs commonly used in ALS, such as riluzole, to the brain. To overcome these limitations and increase brain targeting, riluzole-loaded nanostructured lipid carriers (NLC) were prepared and functionalized with lactoferrin (Lf), facilitating transport across the BBB by interacting with Lf receptors expressed in the brain endothelium. NLC were characterized with respect to their physicochemical properties (size, zeta potential, polydispersity index) as well as their stability, encapsulation efficiency, morphology, in vitro release profile, and biocompatibility. Moreover, crystallinity and melting behavior were assessed by DSC and PXRD. Nanoparticles exhibited initial mean diameters between 180 and 220 nm and a polydispersity index below 0.3, indicating a narrow size distribution. NLC remained stable over at least 3 months. Riluzole encapsulation efficiency was very high, around 94–98%. FTIR and protein quantification studies confirmed the conjugation of Lf on the surface of the nanocarriers, with TEM images showing that the functionalized NLC presented a smooth surface and uniform spherical shape. An MTT assay revealed that the nanocarriers developed in this study did not cause a substantial reduction in the viability of NSC-34 and hCMEC/D3 cells at a riluzole concentration up to 10 μM, being therefore biocompatible. The results suggest that Lf-functionalized NLC are a suitable and promising delivery system to target riluzole to the brain