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The members of Cystus genus are perenial shrubs with a well-established use in traditional medicine. Among these, C. creticus is the most popular, herbal preparations obtained from its aerial parts being recognized as antimicrobial, antitumor and anti-inflammatory agents. The present study aimed to evaluate phytochemical profile and bioactive potential of aqueous and hydroethanolic extracts of C. creticus aerial parts harvested from two different areas of Rhodes island. LC-DAD-ESI/MSn analysis revealed the presence of myricetin and quercetin glycosides as main compounds, especially in aqueous extracts, being probably responsible for their enhanced antioxidant and antimicrobial potential. On the other side, hydroethanolic preparations exerted a strong anti-inflammatory and anti-biofilm activity. Our findings suggest that the use of solvents with intermediate polarity can assure the best recovery of bioactive compounds from C. creticus, increasing the extraction yield for other non-phenolic compounds which can enhance therapeutic potential of the extract through a synergistic action. This work was supported by a grant of the Romanian Ministry of Education and Research, CNCS–UEFISCDI, project number PN-III-P2-2.1-PED-2019-5360. This research was funded by the Serbian Ministry of Education, Science and Technological Development (Contract No. 451–03–9/2021–14/200007). The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) and FEDER under Programme PT2020 for financial support to CIMO (UID/AGR/00690/2020); the national funding by FCT, P.I., through the institutional scientific employment program-contract for LB, RC, and ÂF contract. info:eu-repo/semantics/publishedVersion

Medicinal and aromatic plants (MAPs) are an important source of natural products, which have gained importance as alternatives in the prevention and treatment of diseases, besides being used as raw material for various industries [1]. Lavandula angustifolia L. is one of the most famous aromatic and medicinal plants [2]. Due to its composition, the essential oil of L. angustifolia is reported to have analgesic, sedative, antiseptic, diuretic, cytotoxic, anti-inflammatory, and antioxidant properties [3]. In this sense, the lavender essential oil was obtained by two different methodologies, conventional hydrodistillation by clevenger apparatus according to the European Pharmacopoeia, and non-conventional, microwave-assisted hydrodistillation, in which an experimental design was carried out to optimize the process. The optimal point was obtained by a Response Surface Methodology (RSM), using three variables, namely, Time (minutes),Temperature (ºC), and Power (W) to obtain high contents of linalool and linalool acetate, for which biological activities, such as antimicrobial, have been described. The oils obtained were analyzed by gas chromatography coupled to mass spectrometry, verifying the predominance of oxygenated monoterpenes (79.5-86.4%), with linalool (30.2-29.99%) and linalool acetate (25.6-37.7%) as the main compounds. Antioxidant activity was evaluated through three in vitro methodologies, namely reducing power (RP), 2,2-diphenyl-1 picrylhydrazyl (DPPH) radical-scavenging activity and cellular antioxidant activity (CAA). Furthermore, cytotoxicity activity was evaluated by the sulforhodamine B method, and antimicrobial activity was tested by the microdilution method.The results obtained show that lavender essential oil exhibits an excellent antioxidant, anti-inflammatory and antimicrobial activity, with particularly interesting results for fungistatic activity. Among the obtained essential oils, the microwave extracts rich in linalool and the extract obtained by clevenger hydrodistillation showed the best results for DPPH and PR assays, while no significant differences were obtained for the cellular antioxidant activity assay and anti-inflammatory activity. The extract obtained by clevenger showed better performance in antimicrobial activity. The oils showed cytotoxic activity for liver cells at the maximum concentration tested, and no toxicity was observed for the concentrations presenting anti-inflammatory activity. The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) for financial support through national funds FCT/MCTES (PIDDAC) to CIMO (UIDB/00690/2020 and UIDP/00690/2020) and SusTEC (LA/P/0007/2021); national funding by FCT, P.I., through the institutional and individual scientific employment program-contract for L. Barros, R.C. Calhelha and Rafael Mascoloti Spréa (2020.08092.BD). S.A. Heleno thanks FCT for her individual employment program–contract (CEECIND/03040/2017). info:eu-repo/semantics/publishedVersion

Tripleurospermum inodorum (L.) Sch. Bip. (Asteraceae), scentless mayweed, is a common weed species, but despite its abundancy it is still underexplored in the terms of its bioactivities. The aim of the study was to explore the bioactivities of scentless mayweed methanolic extract including antimicrobial, antibiofilm, antioxidant, cytotoxic, and antiinflammatory activity and to provide deeper insight into its chemical composition. It was shown that T. inodorum inhibits growth of human pathogenic microorganisms with minimal inhibitory concentration (MIC) in range 3-12 mg/mL, with the lowest MIC noted for the species Candida albicans 475/15, C. albicans 17/15, C. parapsilosis ATCC 22019, Pseudomonas aeruginosa (IBRS P001), Yersinia enterocolitica (ATCC 23715), and Klebsiella pneumoniae (ATCC 13883). Its biofilm potential was promising - application of plant extract (1.5 and 3 mg/mL) reduced C. albicans 475/15 biofilm biomass and biofilm cell viability for more than 50%. Antioxidant assays suggested the plants antioxidant potential at 442.2 mmol GAE (gallic acid equivalent)/100 mg DW (dry weight) as in ABTS􀀀+ assay, 277.8 mmol GAE/100 mg DW in DPPH∙, and 204.4 mmol GAE/100 mg DW as recorded in the FRAP assay. Wide cytotoxic potential of the extract was noticed for the range of cells: AGS (gastric adenocarcinoma, GI50 198.7 μg/mL), CaCo2 (colorectal adenocarcinoma, GI50 278.2 μg/mL), MCF-7 (breast adenocarcinoma, GI50 265.7 μg/mL), NCI-H460 (lung carcinoma, GI50 62.9 μg/mL), and VERO (non-tumour cell line, GI50 >400 μg/mL). Likewise, low IC50 (8.4 μg/mL) in the RAW 246.7 anti-inflammatory assay suggests the strong antiinflammatory potential of this weed. The dominant polyphenols in the extract were two apigenin derivatives: apigenin-O-pentoside and apigenin-O-acetylhexoside (5.234 mg/g and 4.929 mg/g extract, respectively). T. inodorum is a rich source of different bioactive polyphenols and its methanolic extract displays a range of bioactive features that should be further examined in order to develop novel herbal medicines This research is funded by the Serbian Ministry of Education, Science and Technological Development (Contract No. 451-03-68/2022-14/200007). Authors are also grateful to Foundation for Science and Technology (FCT, Portugal) for financial support through national funds FCT/MCTES to the CIMO (UIDB/00690/2020). M.I. Dias, R. Calhelha, and L. Barros thank the national funding by FCT, P.I., through the institutional scientific employment program for their contracts. info:eu-repo/semantics/publishedVersion

Although cholesterol is essential for the functioning of the human body, its excess is responsible for causing atherosclerosis, along with several other diseases that affect the bones, liver, hormones, and immune system1. As a natural solution, mushrooms are being studied for their ability to reduce cholesterol due to their richness in molecules capable of reducing both cholesterol absorption and synthesis2,3. Therefore, the present work investigated two mushrooms species (Agaricus bisporus L. and Pleurotus ostreatus L.), aiming at extracting the highest contents of statins and ergosterol. Ultrasound assisted extraction (UAE) optimization was performed followed by quantification of ergosterol and statins of the extracts by HPLC-UV4,5, as well as the effect of the temperature on both extractions (with and without iced bath), since statins are described as sensitive to high temperatures. For A. bisporus, there was no significant differences in the concentration of ergosterol and pravastatin when applying ice bath during the UAE. For P. ostreatus, the ice bath resulted in a 6% decrease in the ergosterol concentration and a 35% increase in pravastatin content, suggesting that the best extraction method to obtain an extract rich in hypocholesterolemic compounds is a cold extraction. For optimization, the P. ostreatus extract revealed the highest amount of pravastatin and ergosterol (63.85 and 23.95 mg/g of extract, respectively), in which the best extraction conditions were 400 W of ultrasonic power, 4 minutes and 44 seconds of extraction time, and 15.2 g/L of solid:liquid ratio, using methanol as solvent, besides using an ultrasound extraction with ice bath. The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) for financial support through national funds FCT/MCTES to the CIMO (UIDB/00690/2020 and UIDP/00690/2020) and SusTEC (LA/P/0007/2021). S. Heleno and M. Carocho thank FCT for their individual employment program–contract (CEECIND/00831/2018, CEECIND/03040/2017), RC Calhelha and L. Barros also thank to the national funding by FCT through the institutional scientific employment program–contract for her contract. info:eu-repo/semantics/publishedVersion

Ruscus aculeatus L. é um subarbusto utilizado na medicina tradicional em várias partes do Mundo, nomeadamente na Europa e na Península Ibérica 1' 1. Tradicionalmente, as partes aéreas são utilizadas como diuréticos e as subterrâneas no tratamento de doenças do trato urinário e como laxante 1' 1. No presente trabalho, a parte aérea, raízes e rizomas de R. aculeatus foram caracterizados quanto às suas propriedades bioativas, nomeadamente: atividade antioxidante, antimicrobiana, anti-inflamatória e citotóxica. O material vegetal de R. aculeatus foi colhido em abril de 2019 em parcelas florestais, em Valpaços, Portugal. Foram preparados extratos hidroetanólicos e aquosos (infusões e decocções) das partes aéreas (caules laminares e ramificações) e das partes subterrâneas (rizomas e raízes). A atividade antioxidante dos extratos foi avaliada mediante a inibição da peroxidação lipídica (TBARS) e da hemólise oxidativa (OxHLIA). A atividade antibacteriana foi avaliada através da técnica de microdiluição, juntamente com o método colorimétrico de deteção rápida com o cloreto de piodonitrotetrazólio (INT). A atividade anti-inflamatória foi avaliada através da inibição da produção de NO utilizando uma linha celular de macrófagos de rato RAW 264.7. As propriedades citotóxicas foram avaliadas utilizando linhas celulares tumorais humanas e numa linha celular não tumoral (células primárias de fígado de porco, PLP2). Todos os extratos revelaram atividade antioxidante e potencial para inibir algumas das bactérias utilizadas. O extrato hidroalcoólico da parte aérea foi o que apresentou melhores resultados na inibição da produção de óxido nítrico seguido pela decocção das raízes e rizoma. Todos os extratos inibiram o crescimento das linhas celulares tumorais, exceto a infusão da parte aérea e os extratos de decocção. Os extratos aquosos da parte aérea e a infusão do extrato de raízes e rizomas, não apresentaram toxicidade para a linha celular PLP2. Este estudo preliminar forneceu resultados inovadores e interessantes em relação às propriedades bioativas desta planta silvestre pouco estudada e explorada. Os autores agradecem à Fundação para a Ciência e Tecnologia (FCT, Portugal) e aos fundos nacionais FCT/MCTES pelo apoio financeiro ao CIMO (UIDB/00690/2020) e pelo financiamento nacional pela FCT, I.P., no âmbito da celebração do contrato-programa de emprego científico de L. Barros, A. Fernandes, C. Pereira e R C. Calhelha. Os autores agradecem ao Fundo Europeu de Desenvolvimento Regional (FEDER) através do Programa Operacional Regional Norte 2020, no âmbito do Projeto Norte-O 1-0 145-FEDER-000042: GreenHealth. info:eu-repo/semantics/publishedVersion

Resumen del trabajo presentado al Transcolab Summit - Trends in grain-based foods, celebrado en Bragança (Portugal) del 23 al 25 de marzo de 2022., This work is part of the R&D project ACRINTAKE (RTI2018–094402-B-I00), financed by MCIN/AEI/10.13039/501100011033/ and "FEDER, A way to make Europe", and partially financed by the Community of Madrid and European funding from the FSE and FEDER programs (project S2018/BAA-4393, AVANSECAL-II-CM). The authors are also grateful to the Foundation for Science and Technology (FCT, Portugal) for financial support by national funds FCT/MCTES to CIMO (UIDB/00690/2020) and to FEDER-Interreg España-Portugal programme for financial support through the Project TRANSCoLAB 0612_TRANS_CO_LAB_2_P.

Adansonia digitata L. é espécie africana com vários usos tradicionais, nomeadamente medicinal e alimentar [1,2]. A polpa do seu fruto, comumente conhecido como mukua, está aprovada como um ingrediente alimentar pela Comissão Europeia e pela “Food and Drug Administration of the United States of America” [1]. Assim o objetivo deste trabalho foi comprovar a potencialidade da polpa da mukua como um alimento funcional através da sua caracterização nutricional, mineral e bioativa. O perfil nutricional foi obtido aplicando metodologias oficiais de análise AOAC [3]. O conteúdo mineral foi determinado por espectrofotometria de absorção atómica e o potencial bioativo foi determinado através da atividade antioxidante pelo ensaio antioxidante celular (CAA) e um ensaio de inibição de substâncias reativas do ácido tiobarbitúrico (TBARS). Além disso, a atividade antimicrobiana foi também determinada, através do método de microdiluição, e a atividade antitumoral foi avaliada em três linhas celulares tumorais humanas: adenocarcinoma gástrico (AGS), carcinoma de mama (MCF-7), células de carcinoma de pulmão (NCI-H460), e a citotoxicidade em culturas de células não tumorais de rim de macaco africano (VERO), através do ensaio da Sulforrodamina B. Relativamente aos resultados obtidos, a polpa de mukua apresentou um teor de humidade muito baixo (11,9 + 0,3 g/100 g ms). Os macronutrientes presentes em maior quantidade foram os hidratos de carbono (89,6 ± 0,2 g/100 g ms), seguidos das proteínas (2,7 ± 0,3 g/100 g ms) e da gordura bruta (1,8 + 0,1 g/100 g ms), representando um valor energético de 386 ± 1 kcal/100 g ms. Quanto ao perfil mineral, a amostra revelou um alto teor em potássio (20,4 ± 0,8 g/100 g ms), estando também presentes minerais como o magnésio (3,28 ± 0,08 g/100 g ms), o cálcio (2,79 ± 0,07 g/100 g ms), o manganês (2,02 ± 0,09 g/100 g ms) e o ferro (1,53 ± 0,09 g/100 g ms). Em relação à atividade antioxidante, a polpa inibiu cerca de 32% da oxidação celular no método CAA e exibiu um valor de EC50 de 23,0 ± 0,1 μg/mL no ensaio de TBARS. Para a atividade antimicrobiana, a polpa apresentou atividade bacteriostática contra a maioria das bactérias testadas. No ensaio de citotoxicidade, a polpa demonstra capacidade de inibir a proliferação celular contra as linhas tumorais testadas, principalmente contra AGS com valores de GI50 de 92 ± 1 μg/mL, sem toxicidade para as células não-tumorais. Este estudo mostra que a polpa de mukua, além de ser nutricionalmente interessante, possui alto teor em minerais, nomeadamente em potássio que é um composto essencial para a bom funcionamento do corpo, estando associado à prevenção de doenças como diabetes, osteoporose e ainda doenças cardiovasculares. Além disso, a polpa da mukua possui alto potencial bioativo, o que indica que o seu consumo pode oferecer benefícios para a saúde, tornando-o num potencial alimento funcional. Os autores agradecem à Fundação para a Ciência e a Tecnologia (FCT, Portugal) e aos fundos nacionais FCT/MCTES (PIDDAC) pelo apoio financeiro ao CIMO (UIDB/00690/2020 e UIDP/00690/2020) e SusTEC (LA/P/0007/2021), pela bolsa de doutoramento (SFRH/BD/145467/2019) de Filipa A. Fernandes, os contratos de Lillian Barros e Ricardo C. Calhelha através do contrato-programa institucional de emprego científico e dos contratos de Sandrina. A. Heleno (CEECIND/03040/2017) e Márcio Carocho (CEECIND/03040/2017) através do contrato-programa individual de emprego científico. Ao FJC2020-045405-I/MCIN/AEI/10.13039/501100011033 e União Europeia “NextGenerationEU”/PRTR” pelo contrato de Tânia C.S.P. Pires. Ao MICINN pelo apoio financeiro Ramón&Cajal a Miguel A. Prieto; ao Governo da Galiza pelo apoio financeiro da Axudas Conecta Peme ao projeto NeuroFood IN852A 2018/58. info:eu-repo/semantics/publishedVersion

A espécie Juniperus communis L., comumente designada “zimbro” em Portugal, é uma conífera, pertencente à familia Cupressaceae e adaptada à baixa disponibilidade de nutrientes no solo. O zimbro foi usado na medicina tradicional para tratar infeções urinárias, dermatites ou como diurético, entre outros, dada a sua composição em bioativos como compostos fenólicos, terpenóides e ácidos orgânicos. Nos últimos anos, vários estudos reportam o seu potencial antimicrobiano, antioxidante, antiinflamatório, efeitos anti-diabéticos, hipocolesterolémicos e ainda a sua ação neuroprotetora, bem como capacidade antiproliferativa contra células cancerígenas e a capacidade de ativar mecanismos hepatorrenais e gastroprotetores [1,2]. No âmbito do presente trabalho, que surge no contexto do projeto BBI-JU BeonNAT, a casca do zimbro foi separada da parte aérea e posteriormente moída à parte da restante biomassa (parte aérea composta por ramos e agulhas) para comparar estas duas frações ao mesmo tempo que se pretende aproveitar todas as partes da planta, promovendo um conceito de economia circular. Depois de moídas, as amostras foram extraídas por maceração e a composição fenólica de cada fração de biomassa foi analisada por HPLC-DAD-ESI/MS, onde 17 compostos foram identificados na casca e 14 compostos na restante biomassa. Enquanto que na casca foram apenas idenditificados flavan-3-ols, na parte aérea a maior parte dos compostos são flavonoides (10 identificados), seguido de 2 flavan-3-ois e ainda 2 ácidos fenólicos, sendo o composto maioritário, nos dois casos, um dímero de procyanidina. Para avaliar a biotividade dos extratos, realizou-se a análise antimicrobiana por microdiluição, utilizando bacterias Gram-positivas e Gram-negativas, isoladas de ambiente clínico e alimentar. A atividade antioxidante foi realizada por duas metodologías distintas: inibição da peroxidação lipídica em tecidos cerebrais de porco (TBARS) e inibição da hemólise oxidativa (OxHLIA), onde o extrato da casca de zimbro se mostrou mais promissor, apresentando um EC50 mais baixo (TBARS: EC50=11.30􀁲0.88 􀁐g/mL e OxHLIA: 14.9±0.5 􀁐g/mL) em comparação com o extracto da parte aérea (TBARS: EC50=25.89􀁲0.62 􀁐g/mL e OxHLIA: 47±1 􀁐g/mL). Por sua vez, a citotoxicidade foi avaliada numa cultura de células não tumorais: PLP2 (cultura primária de células de fígado de porco), em que ambos os extratos apresentaram toxicidade para esta linha celular. No entanto, de acordo com a aplicação-alvo, a toxicidade apresentada pelos extratos deve ser analisada mais profundamente, verificando esta condição em modelos de toxicidade específicos para cada indústria/produto. No geral, tendo em conta a composição química e as bioatividades comprovadas, os extratos quer da casca, quer da parte aérea do zimbro são potenciais matérias-primas para o desenvolvimento de produtos com aplicação na área alimentar, farmacêutica e/ou cosmética. Os autores agradecem à Fundação para a Ciência e a Tecnologia (FCT, Portugal) e aos fundos nacionais FCT/MCTES (PIDDAC) pelo apoio financeiro ao CIMO (UIDB/00690/2020 e UIDP/00690/2020) e SusTEC (LA/P/0007/2021). o contrato de Lillian Barros através do contrato-programa institucional de emprego científico e do contrato de Sandrina. A. Heleno (CEECIND/03040/2017) através do contrato-programa individual de emprego científico. Este projeto recebeu financiamento da Empresa Comum das Indústrias de Base Bio (JU) ao abrigo da convenção de subvenção n.º 887917 BeonNAT. A JU recebe apoio do programa de pesquisa e inovação Horizon 2020 da União Europeia e do Bio Based Industries Consortium. info:eu-repo/semantics/publishedVersion

Peumus boldus L. (boldo) is known for its pharmacological effects associated with the chemical composition of its leaves. The objective of this work was to perform the nutritional, chemical and bioactive characterization of boldo leaves. The nutritional profile (crude fat, protein, ash, fiber, and carbohydrates) was determined using AOAC methods [1], while free sugars were determined by HPLC-RI and fatty acids by GC-FID. Moreover, organic acids were determined by HPLC-DAD, tocopherols by HPLC-fluorescence, and phenolic compounds by HPLC-DAD-ESI/MS. Afterwards, a hydroethanolic extract was prepared by maceration (80:20 v/v, ethanol:water), and the antioxidant potential was evaluated using the thiobarbituric acid reactive substances (TBARS) assay; antimicrobial capacity using the microdilution method against pathogenic microorganisms; cytotoxicity against tumor and non-tumor cell lines using the sulforhodamine B assay, and anti-inflammatory potential using RAW cells. For the nutritional profile results, the highest values were obtained for carbohydrates (78 ± 1 g/100 g dw, of which 48.0 ± 0.2 g/100 g dw corresponded to fiber), followed by proteins (8.2 ± 0.5 g/100 g dw), ash (10.0 ± 0.9 g/100 g dw) and fat (4.24 ± 0. 06 g/100 g dw), having an energetic value of 285 ± 2 kcal. Glucose (6.82 ± 0.03 g/100 g dw), followed by fructose (2.70 ± 0.04 g/100 g dw) and sucrose (1.50 ± 0.10 g/100 g dw) were the detected sugars. Ten fatty acids were found, with three main compounds standing out, namely palmitic (29.56 ± 0.05 %), oleic (19 ± 5 %) and linolenic (17 ± 2 %) acids. Five organic acids were identified, highlighting oxalic (1.26 ± 0.04 g/100 g dw) and quinic (1.24 ± 0.05 g/100 g dw) acids. The vitamin E isoforms γ-tocopherol (15 ± 1 g/100 g dw) and α-tocopherol (5.2 ± 0.3 g/100 g dw) were the ones identified in boldo leaves. Seventeen phenolic compounds were detected, from which proanthocyanidins were the most prominent, especially a proanthocyanidin dimer (34.69 ± 0.91 mg/g), and two proanthocyanidin trimers, 1 (32.12 ± 0.75 mg/g) and 2 (18.59 ± 0.67 mg/g). For the antioxidant potential (TBARS), an EC50 of 36.8 ± 0.2 μg/mL was determined, and for the antimicrobial capacity, lower minimum inhibitory concentrations (MIC) were seen especially against the foodborne bacteria Yersinia enterocolitica (1.25 mg/mL) and Staphylococcus aureus (1.25 mg/mL), as well as the clinical isolates E. coli (2.5 mg/mL), Morganella morganii (2.5 mg/mL), Enterococcus faecalis (2.5 mg/mL) and methicillin-resistant S. aureus (2.5 mg/mL). Boldo leaves showed cytotoxicy against nonsmall lung carcinoma (71 ± 4 μg/mL), gastric adenocarcinoma (159 ± 12 μg/mL), and breast carcinoma (280 ± 9 μg/mL) cells. They also showed anti-inflammatory activity for macrophage cell line (RAW cells) at 145 ± 13 μg/mL. Thus, besides being rich in nutrients, P. boldus revealed the presence of bioactive compounds which can be used for future applications in the food, cosmetic and pharmaceutical industries. The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) for financial support through national funds FCT/MCTES (PIDDAC) to CIMO (UIDB/00690/2020 and UIDP/00690/2020) and SusTEC (LA/P/0007/2021). L. Barros and R.C. Calhelha thank the national funding by FCT through the institutional scientific employment program-contract for her contract, while S. Heleno thank FCT through the individual scientific employment program-contracts (CEECIND/03040/2017). I. Oliveira thanks FCT for her PhD grant (BD/06017/2020). info:eu-repo/semantics/publishedVersion

As borras obtidas na produção de óleo de noz podem ser consideradas como um dos principais subprodutos desta indústria, sendo atualmente reaproveitados como fertilizante ou na alimentação animal [1]. Neste sentido, tendo por objetivo final a valorização deste subproduto, este trabalho consistiu na caracterização química e avaliação da bioatividade das borras geradas na produção de óleo de noz. As borras industriais foram analisadas no que respeita o teor em açúcares livres e tocoferóis usando cromatografia líquida acoplada a detetores de índice de refração (HPLC-RI) e de fluorescência (HPLC-FL), respetivamente. Os ácidos gordos foram determinados por cromatografia gasosa com deteção de ionização de chama (GC-FID), os ácidos orgânicos por cromatografia líquida ultrarrápida acoplada a um detetor de díodos (UPLC-DAD) e os compostos fenólicos por HPLC com deteção DAD e espectrometria de massa (HPLC-DAD-ESI-MS/MS). Adicionalmente, avaliou-se a atividade antioxidante e antimicrobiana do extrato hidroetanólico obtido a partir das borras. A atividade antioxidante foi determinada através de três ensaios in vitro (inibição da peroxidação lipídica por substâncias reativas ao ácido tiobarbitúrico (TBARS) em homogeneizados cerebrais, e atividade antioxidante celular (CAA)) e a atividade antimicrobiana pelo método de microdiluição contra um painel de bactérias e fungos. As borras de nozes apresentaram uma predominância em ácidos gordos polinsaturados, sendo o ácido linoleico o maioritário, apresentando um teor superior a 55%. Em relação aos tocoferóis, foram observadas as isoformas α, γ e δ, sendo o γ-tocoferol o composto predominante (89 mg/100 g). Apenas a sacarose foi identificada no que concerne os açucares livres, e o ácido quinico foi o maioritário (1,06 mg/100 g) entre os cinco ácidos orgânicos identificados. No que respeita a composição fenólica do extrato, observou-se uma predominância de derivados da catequina (flavan-3-ois) e taninos hidrolisáveis. O extrato das borras de nozes demonstrou interessante atividade antioxidante, apresentando ótimos valores de EC50 (TBARS: 32,84 g/mL) e % de inibição (CAA: 90%). Contudo, o extrato não apresentou atividade significativa contra as bactérias e fungos testados. Os resultados deste trabalho demonstram o potencial deste resíduo industrial dada a sua elevada atividade antioxidante e composição em nutrientes e compostos bioativos, podendo o mesmo ser futuramente explorado através da sua incorporação em produtos alimentares, agregando valor à cadeia produtiva de óleo de noz. Os autores agradecem à Fundação para a Ciência e a Tecnologia (FCT, Portugal) e aos fundos nacionais FCT/MCTES (PIDDAC) pelo apoio financeiro ao CIMO (UIDB/00690/2020 e UIDP/00690/2020) e SusTEC (LA/P/0007/2021); à FCT, P.I., através do contrato-programa de emprego científico institucional e individual, os contratos de L. Barros e J. Pinela (CEECIND/01011/2018), respetivamente, e da bolsa de doutoramento de R.M. Spréa (2020.08092.BD); ao programa Internacionalização Portugal2020 o apoio financeiro ao projeto BIOMA (POCI_01_0247_FEDER_046112); ao FEDER através do Programa Operacional Regional Norte 2020, o apoio ao Projeto GreenHealth (Norte-01-0145-FEDER-000042). info:eu-repo/semantics/publishedVersion

The industrial sector is tirelessly searching for more stable and natural colouring food matrices. Considering these demands, the present research investigated the characterization of their most promising compounds in terms of anthocyanin and betalain pigments, followed by the bioactive effect of aqueous extracts obtained from leaves of Tradescantia zebrina Bosse (ETZ) and Alternanthera brasiliana L. (EAB), respectively, with a view to its potential application in the industry. The chemical profile of EAB and ETZ extracts was obtained by HPLC-DAD-ESI/MS. The antioxidant activity of both extracts was evaluated by three different in vitro assays (CAA, reducing power, and DPPH), the antimicrobial activity was tested on a panel of twelve foodborne pathogenic microorganisms and the anti-inflammatory activity was analyzed in rat macrophage cells (RAW 264.7). Finally, cytotoxicity was tested in human tumor cells (MCF-7, NCI-H460, AGS, and CaCo) and toxicity in non-tumor cells (PLP2 and VERO) by the sulforhodamine B method. Two betalain derivatives were found in EAB samples and four anthocyanin derivatives in ETZ samples. The best promising antioxidant activity was showed in DPPH assay (EC50 0.024±0.001 mg/mL, ETZ), followed by reducing power (EC50 0.53±0.01 mg/mL, EAB) and CAA (inhibition: 46% EAB, 62% ETZ, 2000 μg/mL), proving the antioxidant potential of the two extracts. The fungicidal activity of the extracts presented values of ≤ 2 mg/mL, and Bacillus cereus was the most sensitive bacteria, compared to other studied species. Only the EAB extract showed anti-inflammatory activity (IC50 86±3 μg/mL). Promising cytotoxicity results were evidenced, namely for EAB in NCI-H460 tumor cellline (80±8 μg/mL) and for ETZ in AGS tumor cell-line (30±2 μg/mL). None of the extracts showed toxicity for the maximum concentration tested (>400 μg/mL). Thus, it was evident that these plants proved to be a promising alternative for obtaining natural coloring pigments capable of being incorporated into the food industry. The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) for financial support through national funds FCT/MCTES (PIDDAC) to CIMO (UIDB/00690/2020 and UIDP/00690/2020) and SusTEC (LA/P/0007/2021) and for E.O. PIRES JR. grant (2021.05425.BD) and for M.I. Dias, R.C. Calhelha, and L. Barros contracts. This work is funded by the European Regional Development Fund (ERDF) through the Regional Operational Program North 2020, within the scope of Project GreenHealth - Digital strategies in biological assets to improve well-being and promote green health. info:eu-repo/semantics/publishedVersion

As espécies C. sempervirens L. e J. communis L. são amplamente plantadas como arbustos ornamentais em parques e jardins, enquanto C. ladanifer L. é frequentemente encontrada em áreas silvestres. No entanto, estas espécies apresentam um elevado potencial para serem cultivadas em terrenos marginais [1]. Diferentes estudos investigaram a composição química dessas espécies por GC-MS; no entanto, a maioria refere-se ao óleo esencial (OE) obtido por hidrodestilação das folhas de C. ladanifer e C. sempervirens, assim como de bagas de J. communis, sendo escassa a informação sobre outras partes das plantas [2,3]. No âmbito do projeto BBI-JU BeonNAT, o presente estudo teve como objetivo avaliar a composição química de OE extraídos por destilação a vapor da parte aérea dessas três espécies e as suas propriedades bioativas (antioxidante, antibacteriana, citotóxica e anti-inflamatória) tendo como finalidade o desenvolvimento de produtos de base biológica em diferentes indústrias. Assim, cada espécie foi colhida em duas origens diferentes para procurar diferentes quimiotipos associados a diferentes localizações geográficas [4]. Os resultados obtidos sugerem que a colheita dessas espécies em diferentes localizações geográficas interfere no rendimento do OE e respectiva composição química, sendo que o maior teor de terpenos foi observado em C. sempervirens seguido de J. communis. Com exceção de uma das origens de C. ladanifer que apresentou viridiflorol como o composto mais abundante, as três espécies apresentaram em comum o 􀉲-pineno como composto majoritário. Os OEs de C. ladanifer apresentaram potencial antibacteriano, apresentando valores de concentração mínima inibitória (CMI) de 0.3 a 1.25 mg/mL. Relativamente às restantes bioatividades, o OE de C. ladanifer, revelou uma inibição da oxidação de 83%, enquanto que o OE de J. communis apresentou citotoxicidade na linhagem celular MCF-7, e os OEs C. sempervirens e C. ladanifer exibiram o maior potencial na linha celular NCI-H460. Qualquer um dos óleos apresentou actividade antiinflamatória, destacando-se o maior potential na especie de C. sempervirens (IC50: 11-14 􀁐g/mL), seguido do OE de C. ladanifer (IC50: 19-21 􀁐g/mL) e J. communis (IC50: 24-85 􀁐g/mL). As espécies avaliadas revelaram ser uma fonte viável e de baixo custo de OEs que podem ser utilizados para o desenvolvimento de produtos de base biológica em diferentes industrias. Os autores agradecem à Fundação para a Ciência e a Tecnologia (FCT, Portugal) e aos fundos nacionais FCT/MCTES (PIDDAC) pelo apoio financeiro ao CIMO (UIDB/00690/2020 e UIDP/00690/2020) e SusTEC (LA/P/0007/2021). o contrato de Lillian Barros através do contrato-programa institucional de emprego científico e do contrato de Sandrina. A. Heleno (CEECIND/03040/2017) através do contrato-programa individual de emprego científico. Este projeto recebeu financiamento da Empresa Comum das Indústrias de Base Bio (JU) ao abrigo da convenção de subvenção n.º 887917 BeonNAT. A JU recebe apoio do programa de pesquisa e inovação Horizon 2020 da União Europeia e do Bio Based Industries Consortium. info:eu-repo/semantics/publishedVersion

Na Europa, quase metade da produção de frutas e hortaliças é perdida ou desperdiçada, principalmente nas etapas de processamento, através da geração de grandes quantidades de cascas. info:eu-repo/semantics/publishedVersion

Eucalyptus is a large genus of tall evergreen plants belonging to the Myrtaceae family, being also one of the most important trees due to its several uses, specially of timber, pulp and essential oil. The demand of Eucalyptus sp. essential oil has significantly increased as it has been approved as a natural additive. It is widely used in food, flavor, pharmaceutical, and perfumery industries, thanks to its many biological properties, including antibacterial, antifungal, analgesic and antiinflammatory ones. Essential oils have been under intensive research, mainly regarding their bioactive properties (antioxidant, fungitoxic, anti-viral, anti-inflammatory, antimicrobial, among others). Owing to these properties they are potentially interesting for diverse industries including the food industry since one of its main problems concerns pathogenic microorganisms and associated toxins that are responsible for food spoilage. Although the application of essential oils in the food industry may have some limitations, such as impact on the organoleptic properties and low solubility, different delivery strategies such as nanoencapsulation, active packaging and coatings are promising technologies that may overcome these issues without compromising nutritional properties in food systems. info:eu-repo/semantics/publishedVersion

A soja (Glycine max) tem desempenhado um papel relevante na alimentação humana do século 21, não só através de produtos à base do grão, como pelo seu óleo, mas também pela sua utilização na produção de bebida vegetal [1]. Esta última tem-se tornado cada vez mais popular, aumentando a quantidade de grão de soja utilizado e, consequentemente, a quantidade de resíduos gerados. Por forma a contribuir para a implementação de uma economia circular, este trabalho teve como objetivo a caracterização química e nutricional de subprodutos provenientes da indústria de produção de bebidas vegetais de soja, nomeadamente okara e casca de soja. A composição proximal dos resíduos foi determinada pelos métodos oficiais da AOAC, os açúcares livres, tocoferóis e ácidos orgânicos por cromatografia líquida com detetor de índice de refração (HPLC-RI), de fluorescência (HPLC-FL) e de díodos (UPLC-DAD), respetivamente, o perfil de ácidos gordos por cromatografia gasosa com detetor de ionização de chama (GC-FID), e os compostos fenólicos por HPLC acoplado a um DAD em série com espectrometria de massa (HPLC-DAD-ESIMS/ MS). Adicionalmente, procedeu-se à avaliação da atividade antimicrobiana face a oito estirpes bacterianas multirresistentes causadoras de doenças alimentares e duas estirpes de fungos contaminantes de alimentos, e da atividade antioxidante pelos ensaios de inibição da peroxidação lipídica (TBARS), inibição de hemólise oxidativa (OxHLIA) e atividade celular antioxidante (CAA). Relativamente ao okara, este subproduto apresentou elevada humidade, sendo o macronutriente predominante as proteínas. Na casca, verificou-se uma predominância de carboidratos e fibras, e baixo teor de humidade. Na fração lipídica, ambos os subprodutos apresentaram uma predominância de ácidos gordos polinsaturados, sendo os maioritários os ácidos oleico e linoleico para o okara e para a casca, respetivamente. Foram detetadas 3 isoformas de Vitamina E, sendo o γ- e δ-tocoferol identificados no Okara, e o α- e δ-tocoferol na casca do grão de soja, verificando-se uma predominância de δ-tocoferol em ambos os subprodutos. O ácido oxálico foi o único ácido orgânico identificado no okara, enquanto o ácido cítrico foi o predominante na casca de soja. Os elagitaninos e isoflavonas foram os compostos fenólicos maioritários na casca dos grãos de soja, sendo as isoflavonas igualmente predominantes no okara. Ambos os extratos apresentaram propriedades antioxidantes satisfatórias, e no geral apresentaram uma inibição superior contra o crescimento de fungos comparativamente a bactérias. Os resultados obtidos demonstram que os subprodutos de soja têm uma composição interessante do ponto de vista nutricional, podendo ser uma fonte de compostos bioativos e um recurso para o desenvolvimento de novos ingredientes funcionais para a indústria alimentar. Os autores agradecem à Fundação para a Ciência e a Tecnologia (FCT, Portugal) e aos fundos nacionais FCT/MCTES (PIDDAC) pelo apoio financeiro ao CIMO (UIDB/00690/2020 e UIDP/00690/2020) e SusTEC (LA/P/0007/2021); à FCT, P.I., através do contrato-programa de emprego científico institucional e individual, os contratos de L. Barros e J. Pinela (CEECIND/01011/2018), respetivamente, e da bolsa de doutoramento de R.M. Spréa (2020.08092.BD); ao programa Internacionalização Portugal2020 o apoio financeiro ao projeto BIOMA (POCI_01_0247_FEDER_046112); e ao FEDER através do Programa Operacional Regional Norte 2020, o apoio ao Projeto GreenHealth (Norte-01-0145-FEDER-000042). info:eu-repo/semantics/publishedVersion

Pumpkin is a vegetable widely consumed around the world, popularly known for its nutritional and pharmacological properties. Some reports in the literature reveal the rich profile of nutrients and bioactive compounds of pumpkin varieties [1,2]. However, the seeds, peels, and fibers resulting from pumpkin processing are still poorly explored by food industry. Considering the alarming scenario of losses and waste during all the food supply chain worldwide [3] and seeking to promote a more sustainable food system, the present study purposed to investigate the by-products of different varieties of pumpkin as a source of extracts with preservative capacity for food application. For that purpose, hydroethanolic extracts from the varieties “Butternut squash”, “Common pumpkin”, and “Kabocha squash” from Portugal, and “Butternut squash”, “Common pumpkin”, and “Musquée de provence” from Algeria, were evaluated in terms of their bioactivities, more specifically the antioxidant, antimicrobial, and cytotoxic capacities. All the samples presented great antioxidant capacity through two based-cell assays, namely the lipid peroxidation inhibition capacity (TBARS) and the anti-hemolytic activity (OxHLIA). Interestingly, the seeds of the common pumpkin from Algeria did not present antihemolytic properties, despite showing the strongest lipid peroxidation inhibition capacity. In fact, the seeds stood out for all the pumpkin varieties in the TBARS assay, while in the OxHLIA assay, the results were quite similar between the type of bioresidues and between the varieties. Moreover, the samples from Portugal showed greater antioxidant capacity than the Algerian ones. Regarding cytotoxicity, the effect of inhibiting non-tumor cell growth was not observed, even at the highest tested concentration of 400 μg/mL. This first validation is of great importance to prevent food safety issues. These preliminary results are the basis for future studies aiming at the valorization of these bioresidues from food industry as a great source of preservative compounds toward the replacement of synthetic additives with natural alternatives allied to health benefits, as also the promotion of a circular economy The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) for financial support through national funds FCT/MCTES to CIMO (UIDB/00690/2020); national funding by FCT, P.I., through the institutional scientific employment program-contract for C. Pereira and L. Barros contracts and A.K. Molina and M.G. Leichtweis PhD grants (2020.06231.BD and 2020.06706.BD, respectively). To FCT, P.I., within the scope of the Project PRIMA Section 2 - Multi-topic 2019: PulpIng (PRIMA/0007/2019). info:eu-repo/semantics/publishedVersion

Currently, there is a great interest in promoting circular economy perspectives for the recovery of value-added bioactive compounds, potentially exploitable as bioactive natural ingredients, from agri-food bioresidues that causes serious environmental problems. The citrus juice industry is a powerful manufacturing industry that generates huge amounts of waste, mainly peels [1-2]. Material & Methods: A hydrodistillation system was used for the extraction of essential oils, polyphenols and organic acids from lemon, clementine and orange peels, subsequently analysed by GC-MS, UPLC-DAD-ESIMSn and UFLC-PDA respectively. Antioxidant, antimicrobial and/or cytotoxic activities were assessed. Results: The aqueous extract was rich in polyphenols such as hesperidin and sinapoil-O-glucosido. It showed antioxidant (IC50 TBARS = 0.98–1.4 mg/mL), antimicrobial (Staphylococcus aureus MIC = 2.5 mg/mL), and cytotoxic (AGS GI50 = 83 ± 4 mg/mL) activities, especially lemon peels. The essential oils recovered from lemon and clementine peels were rich in limonene (63.6-33,7%), nerol (10,9%) and linalool (5,3%), showing the most antioxidant (IC50 DPPH = 2.0 mg/mL) and antimicrobial (Campylobacter jejuni inhibition halo = 2.8 cm) potential. Supported by Madrid/FEDER program [S2018/BAA-4393, AVANSECAL II-CM]; the Spanish Science Ministry [PID2020-114714RB-100], the Complutense University of Madrid through a pre-doctoral grant [CT17/17 - CT18/17], the Foundation for Science and Technology (FCT, Portugal) by national funds FCT/MCTES to CIMO (UIDB/00690/2020); the C.L. Roriz PhD’s grant (SFRH/BD/117995/2016), the contracts of M.I. Dias, R.C. Calhelha, and L. Barros through the institutional scientific employment program-contract and S.A. Heleno (CEECIND/03040/2017) through the individual scientific employment program-contract. info:eu-repo/semantics/publishedVersion

The cereal-based food formulations has evolved in the last decades with the inclusion of alternative cereals, pseudocereals and other ingredients in traditional recipes, which allows current consumers’ needs to be met. The incorporation of ingredients such as seeds, legume flours or different type of sugars in the innovative formulation of biscuits may be particularly desirable from a nutritional and healthy point of view. However, the dough composition, low moisture and thermal treatment applied during baking can promote the formation of chemical process contaminants, such as acrylamide, which is classified as “probably carcinogenic for humans” [1]. The effect of addition of chia seeds, carob flour and coconut sugar on the nutritional and bioactive properties and the formation of acrylamide in wheat flour-based biscuits was investigated. Different biscuits were formulated replacing wheat flour by chia seeds (percentages in the final weight: 5, 10 and 15%) and carob flour (percentages in the final weight: 1, 5 and 10%) and white sugar by coconut sugar. Biscuit were prepared under controlled conditions and baked for 23 minutes at 180 ºC. Nutritional composition (AOAC methods), in vitro bioactivity (antioxidant capacity (TBARs), antibacterial and antifungal activity) and acrylamide content (HPLC-ESI-MS/MS) were determined in both ingredients and biscuits. Results were statistically analyzed using SPSS version 26 (SPSS, Chicago, IL, USA) assuming a level of significance of p < 0.05. Higher percentages of chia seeds, carob flour and coconut sugar in the formula exhibited similar antibacterial and antifungal activity to the control biscuit (100% wheat flour, 100% white sugar) but increased the antioxidant capacity, protein, fiber and, in the case of chia seeds, the polyunsaturated fatty acids content, then resulting in a nutritionally enhanced product. However, levels of acrylamide were also increased, from 113 to 236 μg/kg, when chia seeds were added in a range of 5-15% of the total weight and reaching values up to 351 μg/kg when carob flour was incorporated in a 10% or even to 1030 μg/kg when biscuit containing 10% carob flour were also formulated with coconut sugar. In these cases, acrylamide concentrations exceeded the benchmark levels (350 μg/kg) established for biscuits in the European Regulation 2158/2017 [2]. This study suggests that the reformulation of traditional recipes with innovative ingredients should be carefully considered, not only looking for nutritionally improved recipes but also taking into account possible changes in the toxicological aspects. To this end, it should be recommended to include a risk/benefit evaluation of the control of process contaminants when designing novel biscuit formulas. This work is part of the R&D project ACRINTAKE (RTI2018–094402-B-I00), financed by MCIN/AEI/10.13039/501100011033/ and "FEDER, A way to make Europe", and partially financed by the Community of Madrid and European funding from the FSE and FEDER programs (project S2018/BAA-4393, AVANSECAL-II-CM). The authors are also grateful to the Foundation for Science and Technology (FCT, Portugal) for financial support by national funds FCT/MCTES to CIMO (UIDB/00690/2020) and to FEDER-Interreg Espa a-Portugal programme for financial support through the Project TRANSCoLAB 0612_TRANS_CO_LAB_2_P. info:eu-repo/semantics/publishedVersion

Artemisia annua L. is known for producing artemisinin, a sesquiterpene lactone compound, which is applied to treat of malaria [1]. Its pharmacodynamic and pharmacokinetic characteristics, as well as its strong capacity for Plasmodium elimination, have raised many expectations for this new class of antimalarial drugs [2]. However, this plant has been also used in traditional Chinese medicine to treat of various diseases since ancient times. Recent studies have focused on its extensive biological activities, which makes this plant an excellent candidate to be used in clinical therapies. Thus, this work aimed to evaluate different bioactivities from a hydroethanolic extract of Artemisia annua. The plant extract in ethanol:water (80:20 v/v) was obtained by microwave-assisted extraction. The antioxidant activity was determined by thiobarbituric acid reactive substances (TBARS) assay, showing a potent activity with an IC50 of 10.3 ± 1.0 μg/mL. A. annua L. extract also exhibited antibacterial and antifungal properties, with concentrations ranging from 2.5 to 10 mg/mL. The ethanolic extract was very effective against the four tumor cell lines, presenting GI50 ranging from 21 to 61 μg/mL and excellent anti-inflammatory activity (38 μg/mL). However, it was also cytotoxic against the two normal cells tested (50 and 19 μg/mL). Concluding, despite the excellent biological activities exhibited by A. annua L. extract, it was also demonstrated to be cytotoxic against normal cells. Therefore, further studies are necessary to study how the cytotoxicity of this extract could be reduced without affecting its bioactive properties. The authors are thankful to the Foundation for Science and Technology (FCT, Portugal) for financial support through national funds FCT/MCTES (PIDDAC) to CIMO (UIDB/00690/2020 and UIDP/00690/2020) and SusTEC (LA/P/0007/2020); national funding by FCT, P.I., through the institutional scientific employment program-contract for R.C.C., and L.B. contract. Manuel Ayuso is also grateful for his financial support through the PRIMA project LocalNutLeg (Section 1 2020 Agrofood Value Chain topic 1.3.1). The authors are also thankful to Project GreenHealth - Norte-01– 0145-FEDER-000042. info:eu-repo/semantics/publishedVersion

The 17 goals of sustainable development address several topics, such as: (2) Zero hunger and sustainable agriculture; (9) Industry, Innovation, and Infrastructure; (12) Responsible consumption and production; that are essential for the promotion of the circular economy, product development and conscious production [1]. Fig is a food matrix, cultivated in Portugal and valued by the Portuguese people for consumption in natura, being also used in wines, liqueurs, and jams. As this fruit is very appreciated and consumed, it´s cultivation leads to the production of tons of leaves, usually discarded [2]. Therefore, in the present work, the leaves of five fig varieties (Figure 1), namely Dauphine (Da), Longue d'Aout (La), Pasteliere (Pa), Marseille (Ma) and Bourjassote Noire (Bn), were nutritionally and chemically characterized to detect possible bioactive molecules. The antioxidant and antimicrobial, activities were also analyzed, to provide the food industry with natural additives in alternative to the artificial ones; and at the same time, promote the circular economy. Regarding the nutritional profile of the five leaves, La sample exhibited the highest amount in proteins (18.0±0.6 g/100g dw), while Pa revealed the highest content in fats (2.2±0.1 g/100g dw). The highest moisture content was presented by Da leaves (17.3±0.1 g/100g fw), and for the ashes, La sample was the one that presented the highest value (14.18±0.06 g/100g fw). Concerning the organic acids, these molecules were most abundant in Ma leaves, where it was possible to identify oxalic, malic and citric acids with a total of 139.6±0.4 mg/g dw. For the soluble sugars profiling, in all samples it was possible to identify five sugars, namely, fructose, glucose, sucrose, trehalose, and raffinose in different concentrations; however, Da leaves revealed the higher amount (17±1 g/100g dw). Tocopherols where also analyzed, and in all samples, three of the four isoforms where detected, being Pa sample standing for the predominance of these compounds (4.14±0.05 mg/100 g dw). For the bioactive analysis, different assays were performed, and Pa sample showed the strongest antioxidant potential for the TBARS assay, with an EC50 value of 105±5 mg/mL. For the antimicrobial activity assay, Da leaf extract was the one displaying the best results, by presenting Minimum Inhibitory Concentrations (MIC) ranging from 1.25 to 10 mg/mL against the tested bacterial strains. On the other hand, for the antifungal activity, the samples present very similar profiles, with the exception of the Pa sample, that present the lowest MIC of 5 mg/mL for Aspergillus fumigatus. In general, these leaf extracts can be used in the food industry namely in pastry and bakery products as promising sources of bioactive compounds, and at the same time, this reuse of biowaste promotes circular economy, and reduces the impact of biowaste resulting from the fig industry, thus meeting some of the goals of sustainable development. The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) for financial support through national funds FCT/MCTES to the CIMO (UIDB/00690/2020). S.Heleno and M. Carocho thank FCT for their individual employment program–contract (CEEC-IND/00831/2018, CEECIND/03040/2017), and L. Barros also thanks to the national funding by FCT through the institutional scientific employment program–contract for her contract. the European Regional Development Fund (ERDF) through the Competitiveness and Internationalization Operational Program for financial support to the project 100% Figo (POCI-01-0247-FEDER-064977) and for C. Shirashi PhD grant. info:eu-repo/semantics/publishedVersion

Market challenges are a strong promoter to innovation in the food preservatives segment, especially regarding consumer resistance to the use of artificial additives [1]. Plants belonging to the genus Thymus are traditionally used as spices in folk medicine and are characterized as promising sources of natural additives [2,3]. Thus, the present work aimed to identify and quantify the phenolic compounds and evaluate the bioactive properties of Thymus mastichina L., to validate its application as a natural preservative ingredient to be applied un the bakery and pastry industry. The chemical composition of Thymus mastichina L. aqueous extract was analyzed by chromatographic methods (HPLC-DAD-ESI/MS), followed by the identification of its individual compounds by comparison to literature data and commercial standards. To prove its bioactive properties, different in vitro tests were carried out to test its antioxidant properties (oxidative hemolysis inhibition (OxHLIA), reducing power, and free radical scavenging capacity (DPPH)), antimicrobial activity (evaluated using a panel of six bacteria and six filamentous fungi), anti-inflammatory action (in rat macrophage cells, RAW 264. 7), and cytotoxicity (in human tumor cell lines: MCF-7, breast carcinoma; NCI-H460, lung cancer; AGS, gastric carcinoma; and in non-tumor cells, PLP2 and Vero, by the sulforhodamine B method). Regarding the individual phenolic compounds, 12 distinct compounds were identified, derived from flavonoids and phenolic acids, in which kaempherol-O-hexuronoside stood out as the major compound. The antioxidant activity was the biological activity that stood out, with the extract presenting low EC50 values (0.048 ± 0.002 mg/mL, 0.035 ± 0.001 mg/mL, and 19.0 ± 0.6 μg/mL for DPPH, reducing power, and OxHLIA assays, respectively). In the cytotoxic assay, the extract showed higher efficiency for AGS cell line (59 ± 5 μg/mL), and for the antimicrobial activity, fungicidal (CMF) and bactericidal (CMB) potential was observed with a concentration range of 2 - 4 mg/mL. In general, based on the bioactive properties demonstrated by thyme extract, it can be considered as a natural ingredient with potential application in the food industry, attributing benefits to new food formulations, especially those developed in the bakery and pastry industry. The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) for financial support through national funds FCT/MCTES to CIMO (UIDB/00690/2020), for E.O. PIRES JR. grant (2021.05425.BD), and for the contracts of C. Pereira, M.I. Dias, R.C. Calhelha, and L. Barros through the institutional scientific employment program-contract. To the project Healthy PETFOOD for the contract of C. Caleja (Project Healthy-PETFOOD (POCI-01-0247-FEDER-047073) and to the BEONNAT (BBI 2019-SO1-R1 - 887917) for the contract of E. Pereira. This work is funded by the European Regional Development Fund (ERDF) through the Regional Operational Program North 2020, within the scope of Project GreenHealth - Digital strategies in biological assets to improve well-being and promote green health, Norte-01-0145- FEDER-000042 and by FEDER-Interreg España-Portugal programme for financial support through the project TRANSCoLAB 0612_TRANS_CO_LAB_2_P. This work has been supported by the Ministry of Education, Science and Technological Development of Republic of Serbia (451-03-68/2020-14/200007). s info:eu-repo/semantics/publishedVersion

Ocimum basilicum 'Cinnamon' is an aromatic and medicinal herb belonging to the Lamiaceae family [1]. Cinnamon basil, as it is popularly known, has aroused the interest of the scientific community for presenting significant concentrations of phenolic compounds [2]. Due to the increasing concern that consumers have towards artificial additives, an intensification in the demand for safer and natural preservatives has been observed [3]. In this sense, the present work aimed to identify the phenolic profile of O. basilicum 'Cinnamon' hydroethanolic extract (80:20, v/v) (EOBC) and to study its bioactive properties. The phenolic composition of the extract was evaluated by a chromatographic method: HPLC-DAD-ESI/MS. Seven distinct compounds were identified, including three phenolic acids (hydroxycinnamic acids) and four flavonoids that corresponded to quercetin derivatives. Regarding the bioactive properties, the antioxidant activity was tested by three in vitro assays: oxidative hemolysis inhibition (OxHLIA), reducing power, and free radical scavenging capacity (DPPH); the cytotoxicity was assessed in human tumor cells (MCF-7, breast carcinoma; NCI-H460, lung cancer; and AGS, gastric carcinoma) and in non-tumor cells (PLP2 and Vero) by the sulforhodamine B method; the antimicrobial activity was evaluated against a panel of twelve food pathogens by the microdilution method, where the maximum bactericidal (MBC) and fungicidal (MFC) concentration values were determined. The results showed a great antioxidant activity, with EC50 values of 0.054 ± 0.002 mg/mL (DPPH), 0.079 ± 0.001 mg/mL (reducing power), and 21.4±0.6 μg/mL (OxHLIA). A remarkable activity against AGS carcinoma (GI50 = 48 ± 3 μg /mL) was also observed. The absence of toxicity of the extract was confirmed up to the maximum concentration studied (>400 μg/mL). In biological terms, EOBC extract showed antimicrobial performance in the ranges of 2-4 mg/mL (CMB) and 1-2 mg/mL (CMF) against all tested strains. In conclusion, it can be observed that O. basilicum 'Cinnamon' represents a valuable natural antioxidant and can also be considered a functional ingredient to be introduced into products of the bakery and pastry sector. The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) for financial support through national funds FCT/MCTES to CIMO (UIDB/00690/2020), for E.O. PIRES JR. grant (2021.05425.BD), and for the contracts of C. Pereira, M.I. Dias, R.C. Calhelha, and L. Barros through the institutional scientific employment program-contract. To the project Healthy-PETFOOD for the contract of C. Caleja (Project Healthy-PETFOOD (POCI-01-0247-FEDER-047073) and to the Valor Natural project for the contract of E. Pereira (Mobilized Project Norte-01-0247-FEDER-024479). This work is funded by the European Regional Development Fund (ERDF) through the Regional Operational Program North 2020, within the scope of Project GreenHealth - Digital strategies in biological assets to improve well being and promote green health, Norte-01-0145- FEDER-000042 and by FEDER-Interreg España-Portugal programme for financial support through the project TRANSCoLAB 0612_TRANS_CO_LAB_2_P. This work has been supported by the Ministry of Education, Science and Technological Development of Republic of Serbia (451-03-68/2020-14/200007 info:eu-repo/semantics/publishedVersion

A indústria alimentar tem vindo a ser desafiada no sentido de impulsionar a inovação nos conservantes alimentares, principalmente devido à resistência dos consumidores em relação à utilização de aditivos artificiais [1]. O género Thymus é constituído por aproximadamente 350 espécies distribuídas por todo o mundo [2]. Estas são tradicionalmente usadas como plantas condimentares e medicinais, destacando-se como fontes promissoras de ingredientes bioativos naturais [3]. O presente estudo pretendeu caracterizar a composição fenólica e avaliar as propriedades bioativas de duas espécies de Thymus (T. mastichina L. e T. pulegioides L.) de forma a comprovar o uso potencial como ingrediente conservante natural na indústria alimentar. Os extratos hidroetanólicos (EtOH/H2O - 80:20, v/v) de T. mastichina (ETM) e T. pulegioides L. (ETP) foram obtidos através de extração assistida por calor. Os compostos fenólicos foram identificados e quantificados por HPLC-DAD-ESI/MS, onde o ETP revelou a presença de catorze compostos fenólicos destacando-se o kaempferol como o composto maioritário; e, por sua vez, o ETM revelou a presença de doze compostos sendo o ácido rosmarínico o maioritário. A atividade antioxidante foi avaliada através de quatro ensaios in vitro: capacidade bloqueadora de radicais livres, poder redutor, inibição de peroxidação lipídica e inibição da hemólise oxidativa. A atividade citotóxica foi avaliada em linhas celulares tumorais humanas (carcinoma da mama, pulmão e gástrico e adenocarcinoma colorretal) e não tumorais (linha primária de fígado de porco, linha celular de rim de macaco), utilizando o método da sulforodamina B. A atividade anti-inflamatória foi testada usando uma linha de macrófagos de rato pela capacidade de inibir a produção de NO. Por fim, a atividade antimicrobiana foi avaliada usando um painel de seis bactérias e seis fungos selecionados segundo a sua importância a nível de saúde pública. Os resultados demostraram que ambos os extratos apresentaram desempenho bioativo promissor, sem apresentar toxicidade até à concentração máxima testada (GI50 > 400 μg/mL). O ETM apresentou melhor potencial antioxidante e antimicrobiano, enquanto o ETP se destacou pelo melhor potencial citotóxico. A composição em compostos fenólicos poderá justificar o desempenho bioativo dos extratos analisados. Desta forma, é possível afirmar que plantas do género Thymus surgem como alternativas naturais para a obtenção de extratos bioativos com potencial de aplicação na indústria alimentar como conservantes naturais. Os autores agradecem à Fundação para a Ciência e Tecnologia (FCT, Portugal) pelo apoio financeiro por meio dos fundos nacionais FCT/MCTES ao CIMO (UIDB/00690/2020 e UIDP/00690/2020) e SusTEC (LA/P/0007/2021).; pela bolsa cedida a E. O. Pires Jr (2021.05425.BD) e pelos contratos de trabalho científico institucional e individual de L. Barros, M.I. Dias, R.C. Calhelha e J. Pinela (CEECIND/01011/2018); ao projeto HealthyPETFOOD (POCI-01-0247- FEDER-047073) pelo contrato de C. Caleja; Este trabalho foi financiado pelo Fundo Europeu de Desenvolvimento Regional (FEDER) através do Programa Operacional Regional Norte 2020, no âmbito do Projeto GreenHealth - Estratégias digitais baseadas em ativos biológicos para melhorar o bem-estar e promover a saúde verde, Norte-01- 0145-FEDER-000042. Este trabalho foi também apoiado pelo Ministério da Educação, Ciência e Desenvolvimento Tecnológico da República da Sérvia (451-03-9/2021-14/200007). info:eu-repo/semantics/publishedVersion

The use of plants for medicinal purposes goes back to ancient times. Adansonia digitata L. is a tree native to the African continent with many traditional uses, including medicinal and food. The fruit of this tree is commonly known as mukua, and consists of pulp, fibrous tissue, and seeds. The pulp has been approved as a food ingredient by the European Commission and the Food and Drug Administration. Consumers are increasingly aware of what they consume, combining food and health, being their preference characterized for more natural products with health benefits. Therefore, nutraceutical products have received more attention from the consumer, being highly acceptable, and the first choice for exigent consumers. Thus, the objective of this work is to exploit the nutraceutical potential of mukua pulp, through its nutritional, chemical and bioactive characterization. The nutritional profile analysis, including proteins, crude fat, moisture, ash, carbohydrates, and energy were performed following the AOAC official methods. Free sugars were identified by an HPLC-RI system, organic acids by UFLC-PDA and fatty acids by GC-FID. For the determination of the bioactive potential, the antioxidant activity was verified through the cellular antioxidant assay (CAA) and the inhibition of thiobarbituric acid reactive substances (TBARS). Its antimicrobial potential against food borne bacteria, fungi and clinical bacteria was analysed through the microdilution method. Furthermore, cytotoxicity was analyzed in three tumor cell lines (gastric adenocarcinoma (AGS), breast carcinoma (MCF-7) and non-small cell lung carcinoma (NCI-H460)) and a non-tumoral cell line (non-tumour culture from African green monkey (VERO)) through the Sulphorhodamine B method. Regarding the results, mukua pulp had a very low moisture content (11.9 + 0.3 g/100 g dw). The macronutrients present in greater amounts were carbohydrates (89.6 ± 0.2 g /100 g dw), followed by proteins (2.7 ± 0.3 g/100 g dw) and by crud fat (1.8 + 0.1 g/100 g dw), representing an energy value of 386 ± 1 kcal. Three sugars (fructose, glucose, and sucrose) and three organic acids were quantified (oxalic, citric, and succinic), being fructose (2.3 ± 0.2 g/100 g dw) and citric acid (8.73 ± 0.03 g/100 g dw) the most abundant ones, respectively. Regarding fatty acids, twelve compounds were quantified with greater abundance of oleic (C18:1n9c – 81%) and palmitic (C16:0 – 10%) acids. Concerning the antioxidant activity, the pulp inhibited about 32% of cell oxidation in the CAA method and exhibiting an EC50 value (concentration providing 50% of antioxidant potential) of 23.0 ± 0.1 μg/mL for TBARS assay. For antimicrobial activity, the pulp presented bacteriostatic activity against most of the food bacteria, with stronger inhibition against Yersinia enterocolitica (minimum inhibitory concentration (MIC) = 0.3 mg/mL), and against the clinical bacteria Enterococcus faecalis (MIC - 0.6 mg/mL). Finally, in the cytotoxicity assay, the pulp demonstrates the ability to inhibit cell proliferation against the tested tumor cell lines, especially against AGS with GI50 values of 92 ± 1 μg/mL, with no toxicity for the normal cells. This study shows that the mukua pulp, in addition to being nutritionally interesting, has a high content of citric acid (scientifically proven preservative power) and high content of oleic acid (multiple pharmacological effects). Furthermore, we proved that the pulp of this fruit, which is so consumed on the African continent, has a high bioactive potential, proving to be a promising candidate to the development of a nutraceutical formulation. info:eu-repo/semantics/publishedVersion