ESS - CISA - Posters apresentados em eventos científicos
Permanent URI for this collection
Browse
Recent Submissions
- Potential skin benefits of incorporating Prunus avium Lapins extracts into a commercially available Portuguese India Pale Ale craft beerPublication . Pereira, Maria João; Santos, Diana; Pinho, Cláudia; Oliveira, Ana Isabel; Pereira, Maria JoãoA commercially available Portuguese India Pale Ale craft beer (ALM-IPA) has shown potential skin benefits in a previous study [1]. However, the incorporation of cherry extracts into bottled beers lacks scientific evidence. To evaluate, in vitro, the benefits of incorporating aqueous (ACE) and ethanolic (ECE) cherry extracts into ALM-IPA beer, in terms of antioxidant and photoprotective activity and the viability of human keratinocytes (HaCaT cells). Experimental study, with the incorporation of ACE (infusion, 1:10) and ECE (70%) (1 mg/mL) into ALM-IPA bottles. Total phenolic content (TPC) was determined. The antioxidant potential was assessed using the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), hydrogen peroxide (H202)and iron-reducing antioxidant power (FRAP) assays. The photoprotective potential was estimated by determining the sun protection factor (SPF) and the ultraviolet absorption capacity (UV-AC). The viability of HaCaT cells was assessed using the 3-(4,5-Dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Data were analysed using the one-way ANOVA test and significant differences were considered for p< 0.05. Regarding antioxidant activity, and comparing both extracts, ALM-IPA+ACE presented the lowest value of IC50for ABTS assay (86.16 ± 5.33 μg/mL) and the highest FRAP value (27.58 ± 0.42 μmol of trolox equivalents/g), which is related with the highest TPC observed (15.10 ± 0.16 mg of gallic acid/g). ALM-IPA+ECE presented the lower IC50(43.27 ± 2.14 μg/mL) compared to ALM-IPA+ACE (65.08 ± 1.69 μg/mL) for H202 assay. However, ALM-IPA beer showed higher antioxidant activity (IC50= 55.21 ± 4.68 μg/mL for ABTS; IC50= 23.54 ± 1.53 μg/mL for H202; FRAP = 53.74 ± 1.27 μmol of trolox equivalents/g). Regarding photoprotective potential, both extracts presented photoprotective potential (SPF > 6) [2]. Analyzing the viability of HaCaT cells after incubation with both extracts, ALM-IPA+ACE and ALM-IPA+ECE presented cytotoxicity, for the 24h and 48h incubation period, only for concentrations higher than 100 μg/mL (cell viability > 80%) [3]. In the 24 hincubation period, ALM-IPA cell viability was higher than ALM-IPA+ACE and ALM-IPA+ECE, and generally, ALM-IPA+ECE was superior to ALM-IPA+ACE. More studies are needed regarding the incorporation of plant extracts into commercially available beers, particularly in other stages of brewing or different styles of beer.
- Beer enriched with “Lapins” cherry extracts: antioxidant activity and liver toxicityPublication . Santos, Diana; Pereira, Maria João Sequeira; Oliveira, Ana Isabel; Pinho, Cláudia; Pinho, CláudiaBeer can be considered a functional beverage and integrate innovative ingredients, namely sweet cherries, with different properties, such as antioxidant activity[1,2]. To evaluate the antioxidant activity, in vitro, and liver toxicity, in human hepatocarcinoma cells (HepG2), in beer after incorporation of aqueous (CAE) and ethanolic (CEE) extracts of cherry variety "Lapins". CAEand CEE(1mg/mL) were incorporated into commercial bottles of Imperial Stout beer (IS-N). The total phenolic content(TPC), expressed in mg of gallicacid equivalents (GAE)/g, was determined. The antioxidant capacity was evaluated by the 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) neutralization and hydrogen peroxide (H₂O₂) assays, both expressed in the concentration required to inhibit the activity by 50% (IC50). Also, the ferricreducing antioxidant power (FRAP) assay was performed and expressed in μmol of trolox equivalent (TE)/mg. Cell toxicity was studied in HepG2 cells, with assessment of metabolic activity by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Data were analysed using GraphPad Prism software, and significant differences were considered for p< 0.05.The incorporation of CEEto IS-N beer significantly decreased TPC (4.2±0.1mg GAE/g for IS-N+CEE; 8.3 ± 0.2mg GAE/g for IS-N;p<0.05).There was an increase in the antioxidant capacity by the ABTS assay(IC50= 80.1±1.1μg/mLfor IS-N; IC50= 60.5 ±1.5 μg/mLfor IS-N + CAE; IC50= 48.0 ±0.6 μg/mLfor IS-N + CEE); however, the incorporation of cherry extracts was not promising in the H2O2(27.0±1.5μg/mLfor IS-N; IC50= 58.7 ±0.4 μg/mLfor IS-N + CEE; IC50= 78.8 ±1.6 μg/mLfor IS-N + CAE;) and FRAP (44.4±0.0μmol/gfor IS-N; 42.7 ±0.0μmol/gfor IS-N + CAE; and 39.7 ±0.0μmol/gfor IS-N + CEE) assays. Further more, IS-N+CEE showed greater antioxidant capacity than IS-N+CAE. After the incorporation of cherry extracts, cytotoxicity was observed in concentrations higher than 10 mg/mL (for IS-N + CAE, 24h incubation) and at the concentration of 500mg/mL (for IS-N + CAE and IS-N + CEE, 48h incubation). IS-N + CEE showed the greatest increase in cell viability. The addition of cherry extracts to beer increased the antioxidant capacity by the ABTS assay, while TPC was reduced with the addition of CEE. The incorporation of both extracts showed promising potential, with low cytotoxicity in HepG2.
- Enantioseparationof 3-chloromethcathinone by liquid chromatography at the milligram scalePublication . Langa, Ivan; Ribeiro, Cláudia; Gonçalves, Virgínia; Dias da Silva, Diana; Cravo, Sara; Tiritan, Maria; Dias da Silva, Diana CristinaThe most prominent synthetic cathinone (SCAT)is 3-chloromethcathinone (3-CMC), accounting for 34% and 63.41% of the total seized new psychoactive substances (NPS) in Europe in 2021 and 2022, respectively [1, 2].Over the latest years, since the first identification on the European drug market in September 2014 in Sweden, 3-CMC has gained significant popularity among the younger drug users [3]. Moreover, 3-CMC is chiral and its enantiomers can show different biological activity, highlighting the importance of the enantio selectivity studies in clinical, forensic and ecotoxicological context. The aim of this study was to optimize a chromatographic method for the enantiomeric separation of 3-CMC at the milligram scale for further use in in vitroand ecotoxicity assessments. The enantio separation as well as the enantiomeric purity evaluation of the 3-CMC were performed by liquid chromatography coupled to the ultraviolet-visible detector (UV/Vis), using a CHIRALPAK®AD-H 10x250mm, 5 μm, a semi-preparative column. A Dionex Ultimate 3000 automated fraction collector was used for fractions collection. Data was analyzed by Chromeleon 7.0 software. For method conditions optimization, a solution at 100 μgmL-1of 3-CMC in ethanol with diethylamine was used. The optimized method allowed the separation of the enantiomers of 3-CMCat final concentration of 3.7 mg mL-1, with an enantiomeric purity of 98 % and 95 % for the first and second eluted enantiomer, respectively. The determination of the absolute configuration of theenantiomers is ongoing by electronic circular dichroism. The isolated enantiomers will be used for the enantio selective evaluation of the 3-CMC ecotoxicity.The determination of the absolute configuration of the enantiomers will enable correlating the ecotoxicity of each enantiomer.
- Design of innovative electrochemical genosensors for honey fraud and quality detection by botanical origin authenticationPublication . Morais, Stephanie L.; Castanheira, Michelle; Pereira, Eduarda; Santos, Marlene; Domingues, Valentina F.; Delerue-Matos, Cristina; Barroso, M. Fátima; Santos, MarleneEnsuring food safety has become a concern for companies, consumers and the government alike, due to the increase of fraudulent and/or adulterated food products found in the global market [1,2]. Some common fraudulent strategies include mislabelling a product's geographical origin and blending it with lower-grade substances [3]. Among the frequently adulterated food products there is honey. Honey is a highly sought-after natural food with an elevated environmental, social and commercial value due to its rich nutritional profile and numerous health benefits. However, it is also vulnerable to adulteration [4]. Therefore, it is important to develop an analytical technique that can quickly, cheaply, and easily assess the quality and safety of honeys worldwide. In this work, an electrochemical genosensor for the detection of two plant species: Erica arborea and Castanea sativa in real honey samples was designed and optimized. The first step was the construction of the genosensor. For this, DNA-target probes capable of unequivocally detecting E. arborea and C. sativa DNA were selected and designed. As a sandwich-format strategy was adopted, their complementary probes were then cut into two smaller sequences to which a thiol group (DNA-capture) and a fluorescein (DNA-signalling) were attached. Using chronoamperometry, the enzymatic amplification of the electrochemical signal was achieved with a concentration range of 0.07 to 2.00 nM. These results were then compared to the DNA from certified E. arborea and C. sativa plant leaves, amplified by PCR, and 10 commercial honeys found in local Portuguese markets. The developed genosensor was successfully applied for the detection of the DNA from the extracted plant leaves and commercial honey samples. So, the developed genosensor is a promising cost-effective and innovative analytical method to detect the botanical origin of real honeys.
- Detecting Candida spp. through an innovative genosensorPublication . Castanheira, Michelle; Morais, Stephanie; Lima, Luís; Santos, Marlene; Barroso, M. Fátima; Santos, MarleneCandida spp. is the second most common cause of invasive fungal infection in hematopoietic stem cell transplantation (HSCT) patients and are associated with high mortality rates, ranging from 40 to 90%. Timely and accurate diagnosis is crucial for successful HSCT treatment. Current diagnostic methods, relying on conventional approaches, have limitations. Molecular and serological tests, although accurate, are time demanding and require specialized equipment. Biosensors are promising tools for point-of-care applications due to their low cost, ease of use and fast results. A low-cost electrochemical genosensor was developed for C. albicans detection. The genosensor construction required a DNA oligonucleotide sequence specific to C. albicans. A 90 bp synthetic DNA fragment was selected to identify this species. The sequence was cut into two fragments: a 25 bp DNA-capture probe and a 65 bp DNAsignaling probe. Screen-printed gold electrodes (SPGE) served as the electrochemical transducer. The sensor design included pretreatment, sensing, sandwich hybridization, and electrochemical detection. SPGE were pretreated with ethanol and ultrapure water. The sandwich assay, the DNA-capture probe bonded to the target DNA, then was immobilized on the working electrode overnight. A SAM interface with capture probes and 6-mercapto-1hexanol was used to ensure probe orientation. Sandwich hybridization improved selectivity by binding the target with a fluorescein-labeled probe. The anti-fluorescein antibody was conjugated with horseradish peroxidase, and the oxidized product was detected by chronoamperometry. Preliminary results show that the sensor was able to detect the synthetic of C. albicans DNA with high selectivity and sensitivity. Further studies will be made to enhance sensitivity parameters. The developed biosensor, with high sensitivity and selectivity, could provide a portable, user-friendly, and low-cost tool for monitoring fungal infections in HSCT recipients.
- Fatores que influenciam a prescrição inicial na diabetes mellitus tipo 2: uma revisão sistemáticaPublication . Moreira, Helena; Moreira, Fernando; Jesus, Ângelo; Soares, M. Monteiro; Santos, P.; Moreira, Fernando; Jesus, ÂngeloCompreender os fatores que influenciam os profissionais de saúde nas suas preferências de prescrição na diabetes tipo 2, uma condição heterogénea e complexa, é fundamental para aprimorar a prática clínica. Este conhecimento também contribui para a personalização dos tratamentos de primeira linha, ajustando-os às características individuais ou a subgrupos específicos, com o objetivo de melhorar os resultados em saúde das pessoas com diabetes tipo 2. Identificar os fatores que influenciam a prescrição da metformina enquanto terapêutica de primeira linha, e avaliar os motivos para a sua não prescrição e o seu alinhamento com as recomendações baseadas na evidência. Secundariamente, procuramos explorar os fatores associados à terapia inicial combinada, uma abordagem mais recente e controversa em comparação com a terapia progressiva. Realizou-se uma revisão sistemática na PubMed (Medline), Scopus e Web of Science de estudos observacionais analíticos que avaliassem os fatores associados à prescrição inicial da metformina ou da terapia combinada. A análise da qualidade metodológica dos estudos foi realizada utilizando as ferramentas do Joanna Briggs Institute. Foram incluídos 30 estudos, com 105 variáveis avaliadas. A prescrição inicial da metformina foi fortemente associada à idade da pessoa com diabetes, valor da hemoglobina glicada, índice de massa corporal e presença de nefropatia, enquanto a terapia combinada se encontra associada à hemoglobina glicada e à presença de comorbidades. Verifica-se uma discrepância entre a prática clínica e as recomendações baseadas em evidência. A maioria das variáveis encontradas derivaram de um único estudo tanto na análise da prescrição inicial (62%) da metformina como na opção por terapêutica combinada (72%). Além disso, foram levantadas questões relacionadas à validade interna e externa dos estudos incluídos. Esta revisão sistemática, que oferece uma visão sobre as práticas clínicas em contexto real, indica uma discrepância entre estas e as recomendações baseadas em evidência. Os nossos resultados destacam a necessidade de intervenções nesta área, além de levantar questões quanto aos motivos associados ao perfil de prescrição dos profissionais de saúde.
- P03-10 Synthetic cannabinoids & neuronal senescence: distinctive responses of in vitro models to AMB-FUBINACAPublication . Bravo, R. Roque; Carmo, H.; Silva, J.P.; Carvalho, F.; Silva, Diana Dias da; Dias da Silva, Diana CristinaAmong the array of new psychoactive substances, synthetic cannabinoids (SC) stand out as highly popular among consumers. These substances closely resemble, in terms of their pharmacology, Δ9-tetrahydrocannabinol (THC), cannabis’ main active principle, albeit exhibiting full agonism at the cannabinoid receptors 1 and 2. In light of recent scientific findings suggesting that cannabis use can exacerbate ageing-related parameters, the present work was designed to explore whether SC share similar potential effects. For this purpose, we employed two distinct in vitro models. The first model involved primary hippocampal cultures (PHC), isolated from Wistar rat embryos at embryonic day 18–19; after seeding, cells were kept in culture and exposed to the popular SC AMB-FUBINACA (AMB-FUB) at 1 pM, 1 nM and 1 µM, starting at day-in-vitro (DIV) 3 or DIV7, and perpetuated until DIV21. DMSO at 0.02% was used as the solvent control. At the end of the exposure, ß-galactosidase activity (a common first-line cell senescence biomarker) was assessed using a commercially-available kit. Our findings under these experimental conditions, revealed that PHC exposed to all AMB-FUB concentrations had less ß-galactosidase activity than the control condition (p<0.01, 1 pM; p<0.001, 1 nM and 1 µM). The other in vitro model used herein was the human neuroblastoma cell line SH-SY5Y. Beginning at passage 24, cells were seeded and exposed to 1 nM and 1 µM AMB-FUB. At passages 24 (48h after drug exposure) and 28, samples were collected for the analysis of several senescence-related endpoints, namely ß-galactosidase activity, cell cycle analysis (via flow cytometry, following DNA staining with propidium iodide) and relative telomere length measurement (using qPCR). Surprisingly, no discernible effect of AMB-FUBINACA was observed for any of the endpoints examined. The apparent observed “anti-ageing” effect of AMB-FUB on PHC warrants further investigation. Moreover, the differential reponse observed between the two in vitro models also requires scrutiny, particularly in light of recent published findings suggesting that THC has the potential to increase ß-galactosidase activity. Further experiments will ensue to hopefully shed some light on these interesting results.
- Optimization of the derivatization procedure for the separation of the stereoisomers of 1,3-dimethylamylamine (1,3-DMAA) by gas chromatography - preliminary dataPublication . Almeida, Maria Mexia de; Silva, Diana Dias da; Oliveira, Ricardo Jorge Dinis; Ribeiro, Cláudia; Dias da Silva, Diana Cristina1,3-Dimethylamylamine (1,3-DMAA),also known as methylhexanamine, is a central nervous system stimulant with structural similarities with amphetamines and therefore presenting over-lapping biological and detrimental effects [1]. Despite being banned, the presence of 1,3-DMAA in dop-ing controls and dietary supplements continues to be of significant concern. This molecule has two stere-ogenic centres and thus four stereoisomers [2]. It is widely recognized that enantiomers may exhibit dif-ferent biological activity, including pharmacokinetics, pharmacodynamics, and toxicity. Consequently, the development of analytical methods for enantioselective separation of 1,3-DMAA is crucial for an accurate determination of the risks associated with each of these stereoisomers. To develop an indirect method by gas chromatography coupled to mass spectrometry (GC-MS) for the separation and identification of the stereoisomers of the 1,3-DMAA. 1,3-DMAA was regenerated with sodium hydroxide, extracted with 0.1% triethylamine in hexane and then derivatized using the enantiomeric pure reagent (R)-(-)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride ((R)-MTPA-Cl). Subsequently, the sample was evaporated, reconstituted in anhydrous ethyl acetate, and analyzed by GC-MS. The chroma-tographic conditions were established using a capillary column containing 5% diphenyl-95% dime-thylpolysiloxane (30 m × 0.25 mm × 0.25 μm), an injector temperature set to 280 ºC, with a temperature ramp starting at 140 ºC and increasing up to 215 ºC at a flow rate of 1 mL/min to a total run of 12.32 min. Results: As preliminary data indicate, the derivatization procedure allowed the formation of 4 diastere-omers of 1,3-DMAA. The chromatographic conditions were optimised, allowing for the separation of the four diastereomers within 12 min. Derivatization and chromatographic conditions were established for enantioselective separation of 1,3-DMAA by GC-MS. Further validation of the method will be crucial for understanding the diastereomers' differential pharmacokinetics and pharmacodynamics, and consequently, the perils associated with their presence in food supplement samples.
- Preliminary chemical profile and in vitro pharmacological evaluation of the hallucinogenic plant Diplopterys cabreranaPublication . Garcia, Maria Rita; Dutka, Mykhaylo; Guimarães, Sofia; Andrade, Paula B.; Seabra, Vítor; Diana Dias da Silva; Gomes, Nelson G. M.; Dias da Silva, Diana CristinaFor the last few years, Ayahuasca ceremonies have been gaining popularity in recreational settings in Europe and North America [1]. Similar to Psychotria viridis, Diplopterys cabreranais also suggested to contain the psychoactive compound N,N-dimethyltryptamine, and is therefore used in Aya-huasca rituals for its ability to induce hallucinations, euphoria and entheogenic effects [1-3]. However, while information on the toxic profile of D. cabreranaremains very limited, its acquisition is easily ac-complished by consumers. We aimed to characterize the aqueous extracts of D. cabreranaleaves, mimicking those typically consumed, to identify bioactives that underlie the psychoactive or toxic effects, and evaluate their impact on neuronal function, neurotransmission and radical stress. Chemical characterization was attained by HPLC-DAD. Impact upon neuronal viability was assessed by the MTT assay (up to 1000 μg/mL) in SH-SY5Y neuroblastoma cells. Impact on neuromodulation and neuroinflammation was evaluated through acetylcholinesterase and 5-lipoxygenase inhibition, while an-tiradical properties were assessed by evaluating nitric oxide (•NO) and xanthine oxidase (XO) activity. Inhibition of the α-glucosidase enzyme was also evaluated. Statistical comparisons among groups per-formed by one-way ANOVA followed by Dunnett post hoc test. Preliminary characterization results revealed the presence of several catechin derivates, alongside two apigenin derivates and one tryp-tamine derivate. Cytotoxicity was not verified up to the highest concentration tested. Acetylcholinesterase inhibition was recorded starting at 250 μg/mL, and a concentration-dependent inhibition of 5-lipoxygen-ase was found (IC50=79.77 μg/mL). Concentration-dependent scavenging effects upon •NO and XO inhi-bition were verified at concentrations higher than 1.953 μg/mL and 31.25 μg/mL, respectively. At last, inhibition of α-glucosidase occurred with concentration-dependency and an IC50of 4.78 μg/mL. Conclu-sions:Although antiradical, anti-inflammatory and antidiabetic properties were verified, with no in vitrocytotoxicity being detected, further research is needed to elucidate the underlying mechanisms that might be involved in our preliminary results.
- In vitro and in silico evaluation of psilocybin and psilocin’s interaction with CYP450 enzymesPublication . Brito-da-Costa, Andreia Machado; Carvalho, Mariana; Dinis-Oliveira, Ricardo Jorge; Madureira-Carvalho, Áurea; Sousa, Sérgio F.; Silva, Diana Dias da; Dias da Silva, Diana CristinaPsilocybin is a hallucinogen produced by “magic mushrooms”, being rapidly metabolized in the organism into psilocin [1, 2]. A scientific gap exists regarding the possible interactions between psilocybin/psilocin and CYP450 enzymes. Given their biological importance, and since the binding of drugs to CYP450 enzymes can interfere with the metabolism of other substrates leading to drug-drug interactions, this research topic is of utmost importance. This study aimed to evaluate in silicoand in vitrothe interaction of psilocybin and psilocin with the enzymes CYP2A6/2B6/2D6/2E1/3A4. The in vitroassessment of inhibition was performed using the Vivid®CYP450 screening kits. IC50 was calculated using GraphPad Prism 9.3.0. For in silico assessment, molecular dynamics were per-formed using the PMEMD.cuda module in AMBER16. Calculations were made on the last 100 ns of the trajectory (stable zone) to assess the interaction between enzyme and ligand, namely MMGBSA, per-residue decomposition energy, and hydrogen bonds. Psilocin showed the capacity to be an in-hibitor of CYP2A6/2B6/2D6/2E1/3A4, based on the respective IC50 values (μM) of 2.06, 6.17, 11.89, 6.37, and 2.36. Considering the MMGBSA, higher values were obtained for psilocin, corroborating the stronger binding affinity of this compound. The interaction of psilocybin/psilocin with CYP2A6 is medi-ated by a hydrogen bond established with the protein residue Asn297. Other important residues include Phe107 and Ile366. For CYP2B6, the strong binding of psilocin is mediated by interactions with Ile114, Thr302 (hydrogen bond), and Leu363. For interaction with CYP2D6, the most important residue seems to be Ser304, with which it forms a hydrogen bond; for CYP2E1, key residues include Phe207, Thr303, and Phe478. A strong hydrogen bond is formed between psilocin and CYP3A4 residue Phe304, contrib-uting to the high binding affinity. The results suggest a potential for psilocin to inhibit all enzymes, especially CYP2A6 and CYP3A4.