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- The salivary volatome in breast cancerPublication . Pereira, Jorge A. M.; Taware, Ravindra; Porto-Figueira, Priscilla; Rapole, Srikanth; Câmara, José S.
- Ultrasound-assisted liquid-liquid extraction followed by ultrahigh pressure liquid chromatography for the quantification of major carotenoids in tomatoPublication . Figueira, José A.; Pereira, Jorge A.M.; Porto-Figueira, Priscilla; Câmara, José S.Lycopene and β-carotene, the main carotenoids present in different tomatoes varieties (gordal, cherry, roma and campari) of Solanum lycopersicum L. (Solanaceae), were investigated using ultrasound-assisted liquid-liquid extraction (LLUSAE) followed by ultra-high pressure liquid chromatography with PDA detection (UHPLC-PDA) analysis. Experimental parameters influencing the LLUSAE were optimized using an univariate design, resulting in a 30-min ACN/MeOH extraction by sonication of a lyophilized sample, followed by PSA/C18/MgSO4 clean-up and fast centrifugation before UPLC analysis. Using this greener methodology, high recoveries (above 97%), good linearity (r2 > 0.98) and improved sensitivity, with limits of detection and quantification of 24.0 and 80.0 ng/mL for lycopene and 3.0 and 9.9 ng/mL for β-carotene, respectively, were obtained. This sensitivity is about five times better than previously reported in literature, making LLUSAE/UHPLC-PDA a promising strategy for lycopene and β-carotene quantification in tomato and eventually in other matrices. The carotenoids studied, lycopene and β-carotene, were found at highest concentrations in the gordal tomato variety, followed by cherry, roma and campari (727.1, 342.2, 267.2 and 218.2 μg/g and 80.4, 44.0, 45.7 and 44.0 μg/g for lycopene and β-carotene, respectively). Additionally, an exponential increase of both carotenoids occurs during ripening and mainly in the skin and locular cavity of the gordal variety. These results provide further evidences of the potential of tomatoes as an interesting source of lycopene and β-carotene.
- Antitumor efficacy of doxorubicin-loaded laponite/alginate hybrid hydrogelsPublication . Gonçalves, Mara; Figueira, Priscilla; Maciel, Dina; Rodrigues, João; Shi, Xiangyang; Tomás, Helena; Li, YulinDegradable hybrid hydrogels with improved stability are prepared by incorporating nanodisks of biocompatible laponite (LP) in alginate (AG) hydrogels using Ca2+ as a crosslinker. The Dox‐loaded hybrid hydrogels give a controlled Dox release at physiological environment in a sustained manner. Under conditions that mimic the tumor environment, both the sustainability in the Dox release (up to 17 d) and the release efficiency from LP/AG‐Dox hydrogels are improved. The in situ degradation of these hybrid hydrogels gives rise to nanohybrids that might serve as vehicles for carrying Dox through the cell membrane and diminish the effect of Dox ion‐trapping in the acidic extracellular environment of the tumor and/or in the endo‐lysosomal cell compartments.
- The potential of microextraction techniques for the analysis of bioactive compounds in foodPublication . Pereira, Jorge A. M.; Casado, Natalia; Porto-Figueira, Priscilla; Câmara, José S.For a long time, the importance of sample preparation and extraction in the analytical performance of the most diverse methodologies have been neglected. Cumbersome techniques, involving high sample and solvent volumes have been gradually miniaturized from solid-phase and liquid-liquid extractions formats and microextractions approaches are becoming the standard in different fields of research. In this context, this review is devoted to the analysis of bioactive compounds in foods using different microextraction approaches reported in the literature since 2015. But microextraction also represents an opportunity to mitigate the environmental impact of organic solvents usage, as well as lab equipment. For this reason, in the recent literature, phenolics and alkaloids extraction from fruits, medicinal herbs, juices, and coffee using different miniaturized formats of solid-phase extraction and liquid-liquid microextraction are the most popular applications. However, more ambitious analytical limits are continuously being reported and emergent sorbents based on carbon nanotubes and magnetic nanoparticles will certainly contribute to this trend. Additionally, ionic liquids and deep eutectic solvents constitute already the most recent forefront of innovation, substituting organic solvents and further improving the current microextraction approaches.
- Exploring the potentialities of an improved ultrasound-assisted quick, easy, cheap, effective, rugged, and safe-based extraction technique combined with ultrahigh pressure liquid chromatography-fluorescence detection for determination of Zearalenone in cerealsPublication . Porto-Figueira, Priscilla; Camacho, Irene; Câmara, José S.Zearalenone (ZEA), a secondary metabolite from Fusarium graminearum fungi that invade crops and grow during blooming in moist cool field conditions, is a nonsteroidal oestrogen, biologically very potent, although hardly toxic. Different analytical methods, among which a quick, easy, cheap, effective, rugged, and safe (QuEChERS) strategy, combined with or liquid chromatography, have been proposed for ZEA determination in foods. However, this extraction procedure has the disadvantage of using large amount of organic solvents, partitioning salts and sample volume, which limits the technique application on the fields where the sample amount is critical. Therefore in this work an improved sample preparation step based on the original QuEChERS (O-QuEChERS) approach, the μ-QuEChERS, combined with ultrasound-assisted extraction (USAE) and ultrahigh pressure liquid chromatography (UHPLC) analysis with fluorescence detection (FLR) is proposed for the sensitive and high-throughput quantification of ZEA in cereals. The proposed analytical strategy, μ-QuEChERSUSAE, uses up to 32 times lower amounts of partitioning salts, lower sample amount and lower extraction solvents, in comparison with O-QuEChERS technique. The performance of the analytical approach was assessed by studying the selectivity, specificity, limits of detection and quantification, linear dynamic range, matrix effect and precision. Good linearity (r(2)>0.99) was achieved for ZEA, and limits of detection (LOD=3.4μgkg(-1)) and limits of quantification (LOQ=4.7μgkg(-1)) were found below the tolerance levels set by European Commission. Good recoveries were obtained with different spiked concentrations, ranged from 80.2% to 109.7%, with relative standard deviations (RSD) lower than 5.0%. The comparison of the analytical performance of the proposed μ-QuEChERSUSAE with O-QuEChERS showed the powerful ability of the proposed strategy since it gives comparable results but using lower amounts of sample, partitioning salts and volume of the organic solvent, revealing of great potential and improvement of the proposed method.
- A non-invasive approach to explore the discriminatory potential of the urinary volatilome of invasive ductal carcinoma of the breastPublication . Taunk, Khushman; Taware, Ravindra; More, Tushar H.; Porto-Figueira, Priscilla; Pereira, Jorge A. M.; Mohapatra, Rajkishore; Soneji, Dharmesh; Câmara, José S.; Nagarajaram, H. A.; Rapole, SrikanthWorldwide, breast invasive ductal carcinoma (IDC) accounts for the majority of the reported cases of this form of cancer. IDC effective management, as for any form of cancer, would greatly benefit from early diagnosis. This, however, due to various socio-economic reasons, is very far for the reality in developing countries like India, where cancer diagnosis is often carried out at late stages when disease management is troublesome. With the present work, we aim to evaluate a simple analytical methodology to identify a set of volatile organic compounds (VOCs) in urine samples, as a biosignature for IDC. Using solid-phase microextraction followed by gas chromatography/mass spectrometry, a panel of 14 urinary VOCs was found to discriminate IDC (n = 65) from a healthy control (HC) group (n = 70) through multivariate statistical treatments. Furthermore, metabolic pathway analysis revealed various dysregulated pathways involved in IDC patients hinting that their detailed investigations could lead to novel mechanistic insights into the disease pathophysiology. In addition, we validated the expression pattern of five of these VOCs namely 2-ethyl-1-hexanol, isolongifolenone, furan, dodecanoic acid, 2-methoxy-phenol in another external cohort of 59 urinary samples (IDC = 32 and HC = 27) and found their expression pattern to be consistent with the primary sample set. To our knowledge, this is the first study exploring breast IDC volatome alterations in Indian patients.
- Urinary Volatomic Expression Pattern: Paving the Way for Identification of Potential Candidate Biosignatures for Lung CancerPublication . Taunk, Khushman; Porto-Figueira, Priscilla; Pereira, Jorge A. M.; Taware, Ravindra; Costa, Nattane Luíza da; Barbosa, Rommel; Rapole, Srikanth; Câmara, José S.The urinary volatomic profiling of Indian cohorts composed of 28 lung cancer (LC) pa tients and 27 healthy subjects (control group, CTRL) was established using headspace solid phase microextraction technique combined with gas chromatography mass spectrometry methodology as a powerful approach to identify urinary volatile organic metabolites (uVOMs) to discriminate among LC patients from CTRL. Overall, 147 VOMs of several chemistries were identified in the intervention groups—including naphthalene derivatives, phenols, and organosulphurs—augmented in the LC group. In contrast, benzene and terpenic derivatives were found to be more prevalent in the CTRL group. The volatomic data obtained were processed using advanced statistical analysis, namely partial least square discriminative analysis (PLS-DA), support vector machine (SVM), random forest (RF), and multilayer perceptron (MLP) methods. This resulted in the identification of nine uVOMs with a higher potential to discriminate LC patients from CTRL subjects. These were furan, o-cymene, furfural, linalool oxide, viridiflorene, 2-bromo-phenol, tricyclazole, 4-methyl-phenol, and 1-(4-hydroxy-3,5-di-tert-butylphenyl)-2-methyl-3-morpholinopropan-1-one. The metabolic pathway analysis of the data obtained identified several altered biochemical pathways in LC mainly affecting glycolysis/gluconeogenesis, pyruvate metabolism, and fatty acid biosynthesis. Moreover, acetate and octanoic, decanoic, and dodecanoic fatty acids were identified as the key metabolites responsible for such deregulation. Furthermore, studies involving larger cohorts of LC patients would allow us to consolidate the data obtained and challenge the potential of the uVOMs as candidate biomarkers for LC.
- Exploring a volatomic-based strategy for a fingerprinting approach of Vaccinium padifolium L. berries at different ripening stagesPublication . Porto-Figueira, Priscilla; Figueira, José A.; Berenguer, Pedro; Câmara, José S.The effect of ripening on the evolution of the volatomic pattern from endemic Vaccinium padifolium L. (Uveira) berries was investigated using headspace-solid phase microextraction (HS-SPME) followed by gas chromato graphy/quadrupole-mass spectrometry (GC–qMS) and multivariate statistical analysis (MVA). The most sig nificant HS-SPME parameters, namely fibre polymer, ionic strength and extraction time, were optimized in order to improve extraction efficiency. Under optimal experimental conditions (DVB/CAR/PDMS fibre coating, 40 °C, 30 min extraction time and 5 g of sample amount), a total of 72 volatiles of different functionalities were isolated and identified. Terpenes followed by higher alcohols and esters were the predominant classes in the ripening stages – green, break and ripe. Although significant differences in the volatomic profiles at the three stages were obtained, cis-β-ocimene (2.0–40.0%), trans-2-hexenol (2.4–19.4%), cis-3-hexenol (2.5.16.4%), β-myrcene (1.9–13.8%), 1-hexanol (1.7–13.6%), 2-hexenal (0.7–8.0%), 2-heptanone (0.7–7.7%), and linalool (1.9–6.1%) were the main volatile compounds identified. Higher alcohols, carboxylic acids and ketones gradually increased during ripening, whereas monoterpenes significantly decreased. These trends were dominated by the higher alcohols (1-hexanol, cis-3-hexenol, trans-2-hexenol) and monoterpenes (β-myrcene, cis-β-ocimene and trans-β ocimene). Partial least squares regression (PLSR) revealed that ethyl caprylate (1.000), trans-geraniol (0.995), ethyl isovalerate (−0.994) and benzyl carbinol (0.993) are the key variables that most contributed to the successful differentiation of Uveira berries according to ripening stage. To the best of our knowledge, no study has carried out on the volatomic composition of berries from endemic Uveira.
- Evaluation of the Health-Promoting Properties of Selected FruitsPublication . Figueira, José A.; Porto-Figueira, Priscilla; Berenguer, Cristina; Pereira, Jorge A. M.; Câmara, José S.In this study, the health-promoting benefits of different fruits grown in Madeira Island, namely lemon (Citrus limon var. eureka), tangerine (Citrus reticulata var. setubalense), pitanga (Eugenia uniflora var. red), tomato (Solanum lycopersicum var. gordal) and uva-da-serra, an endemic blueberry (Vaccinium padifolium Sm.), were investigated. The phenolic composition (total phenolics and total flavonoids content) and antioxidant capacity (assessed through ABTS and DPPH assays) were measured revealing a high phenolic potential for all fruits, except tomato, while uva-da-serra is particularly rich in flavonoids. In relation to the antioxidant capacity, the highest values were obtained for pitanga and uva-da-serra extracts. The bioactive potential was also assessed through the ability of the extracts to inhibit digestive enzymes linked to diabetes (α-amylase, α- and β glucosidases) and hypertension (angiotensin-converting enzyme, ACE). The results obtained point to a very high bioactive potential with the selected samples exhibiting very important ACE anti enzymatic capacities. A statistical analysis of the obtained data reveals a very strong correlation between ABTS and TPC, and a strong contribution of the fruit polyphenols for enzyme inhibition, and thus, presenting high antihypertensive and antidiabetic capacities. Overall, the results obtained clearly show a high bioactive potential of the selected fruits that should be further studied, in terms of specific phenolic composition. Moreover, these results strongly support the valorisation of pitanga seeds usually discarded as a waste, and uva-da-serra, an endemic and wild bush, as potential bioresources of bioactive compounds with impact in human diet.
- Tangerines cultivated on Madeira Island: a high throughput natural source of bioactive compoundsPublication . Figueira, José A.; Porto-Figueira, Priscilla; Pereira, Jorge A. M.; Câmara, José S.Tangerines (Citrus reticulata) are popular fruits worldwide, being rich in many bioactive metabolites. The setubalense variety cultivated on Madeira Island has an intense aroma easily distinguishable from other tangerines, being traditionally used to enrich several foods and beverages. Nonetheless, setubalense volatile composition has never been characterized, and we aimed to unveil the bioactive potential of peels and juices of setubalense tangerines and compare them with the murcott variety grown in Portugal mainland. Using headspace solid-phase microextraction coupled to gas chromatography mass spectrometry (HS-SPME/GC-MS), we identified a total of 128 volatile organic metabolites (VOMs) in the juice and peels, with d-limonene, γ-terpinene, β-myrcene, α- and β-pinene, o-cymene, and terpinolene, the most dominant in both cultivars. In contrast, setubalense juices are richer in terpenes, many of them associated with health protection. Discriminant analysis revealed a pool of VOMs, including β-caryophyllene and E-ocimene, with bioactive properties able to differentiate among tangerines according to variety and sample type (peel vs. juice). This is the first report on the volatile composition of setubalense tangerines grown on Madeira Island revealing that its pungent aroma is constituted by secondary metabolites with specific aroma notes and health properties. This is strong evidence of the higher nutraceutical value of such fruit for the human diet.
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