Chemical Profile of Lipophilic Fractions of Different Parts of Zizyphus lotus L. by GC-MS and Evaluation of Their Antiproliferative and Antibacterial Activities

Zizyphus lotus L. is a perennial shrub particularly used in Algerian folk medicine, but little is known concerning the lipophilic compounds in the most frequently used parts, namely, root bark, pulp, leaves and seeds, which are associated with health benefits. In this vein, the lipophilic fractions of these morphological parts of Z. lotus from Morocco were studied by gas chromatography–mass spectrometry (GC–MS), and their antiproliferative and antimicrobial activities were evaluated. GC– MS analysis allowed the identification and quantification of 99 lipophilic compounds, including fatty acids, long-chain aliphatic alcohols, pentacyclic triterpenic compounds, sterols, monoglycerides, aromatic compounds and other minor components. Lipophilic extracts of pulp, leaves and seeds were revealed to be mainly composed of fatty acids, representing 54.3–88.6% of the total compounds detected. The leaves and seeds were particularly rich in unsaturated fatty acids, namely, (9Z,12Z)-octadeca-9,12-dienoic acid (2431 mg kg−1 of dry weight) and (9Z)-octadec-9-enoic acid (6255 mg kg−1 of dry weight). In contrast, root bark contained a high content of pentacyclic triterpenic compounds, particularly betulinic acid, accounting for 9838 mg kg−1 of dry weight. Root bark extract showed promising antiproliferative activity against a triple-negative breast cancer cell line, MDA-MB-231, with a half-maximal inhibitory concentration (IC50 ) = 4.23 ± 0.18 µg mL−1 of extract. Leaf extract displayed interesting antimicrobial activity against Escherichia coli, methicillin-sensitive Staphylococcus aureus and Staphylococcus epidermis, presenting minimum inhibitory concentration (MIC) values from 1024 to 2048 µg mL−1 of extract. Our results demonstrate that Zizyphus lotus L. is a source of promising bioactive components, which can be exploited as natural ingredients in pharmaceutical formulationspublishe

Lysine-PEGylated Cytochrome C with Enhanced Shelf-Life Stability

Cytochrome c (Cyt-c), a small mitochondrial electron transport heme protein, has been employed in bioelectrochemical and therapeutic applications. However, its potential as both a biosensor and anticancer drug is significantly impaired due to poor long-term and thermal stability. To overcome these drawbacks, we developed a site-specific PEGylation protocol for Cyt-c. The PEG derivative used was a 5 kDa mPEG-NHS, and a site-directed PEGylation at the lysine amino-acids was performed. The effects of the pH of the reaction media, molar ratio (Cyt-c:mPEG-NHS) and reaction time were evaluated. The best conditions were defined as pH 7, 1:25 Cyt-c:mPEG-NHS and 15 min reaction time, resulting in PEGylation yield of 45% for Cyt-c-PEG-4 and 34% for Cyt-c-PEG-8 (PEGylated cytochrome c with 4 and 8 PEG molecules, respectively). Circular dichroism spectra demonstrated that PEGylation did not cause significant changes to the secondary and tertiary structures of the Cyt-c. The long-term stability of native and PEGylated Cyt-c forms was also investigated in terms of peroxidative activity. The results demonstrated that both Cyt-c-PEG-4 and Cyt-c-PEG-8 were more stable, presenting higher half-life than unPEGylated protein. In particular, Cyt-c-PEG-8 presented great potential for biomedical applications, since it retained 30-40% more residual activity than Cyt-c over 60-days of storage, at both studied temperatures of 4 °C and 25 °C.publishe

Chemical characterisation, antioxidant and antibacterial activities of Pinus pinaster Ait. and Pinus pinea L. bark polar extracts: prospecting Forestry by-products as renewable sources of bioactive compounds

Agroforestry by-products have gained rising attention in recent years as they represent inexpensive and abundant raw materials that are a source of added-value chemicals, e.g., for food and pharmaceutical applications, as well as for bioenergy generation. Pinus pinaster Ait. bark extracts are consumed worldwide for their cardiovascular benefits, whilst the health potential of Pinus pinea L. bark has not yet been deeply exploited. Therefore, this study highlights the chemical characterisation of Portuguese P. pinaster Ait. and P. pinea L. bark polar extracts, via ultra-high performance liquid chromatography-diode array detection-tandem mass spectrometry (UHPLC-DAD-MSn) analysis, and their antioxidant and antibacterial activities. Quinic acid, an A-type procyanidin dimer isomer, protocatechuic acid, and quercetin were identified for the first time as P. pinea L. bark components. Moreover, this bark demonstrated a higher total content of identified polar compounds than P. pinaster Ait. bark, with quinic acid being the most abundant compound identified. Regarding antioxidant activity, the pine bark polar extracts exhibited strong reducing power and 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2-azinobis-(3-ethyl-benzothiazoline-6-sulfonic acid (ABTS) radical scavenging effects compared to natural antioxidants. Moreover, the bactericidal actions of pine bark extracts were shown against Staphylococcus aureus and Escherichia coli at a 3.13–25 mg mL−1 range. Globally, these promising insights can boost the sustainable exploitation of P. pinea L. bark, as already occurs with P. pinaster Ait. bark, for the food and biomedical fields.publishe

Conductive polysaccharides-based proton-exchange membranes for fuel cell applications: the case of bacterial cellulose and fucoidan

Conductive natural-based separators for application in polymer electrolyte fuel cells (PEFCs) were fabricated by combining a bacterial polysaccharide, i.e. bacterial cellulose (BC), and an algae sulphated polysaccharide, i.e. fucoidan (Fuc). The diffusion of fucoidan aqueous solution containing a natural-based cross-linker, viz. tannic acid, into the wet BC nanofibrous three-dimensional network, followed by thermal cross-linking, originated fully bio-based proton exchange membranes (PEMs). The PEMs present thermal-oxidative stability in the range of 180-200 °C and good dynamic mechanical performance (storage modulus ≥ 460 MPa). Additionally, the BC/Fuc membranes exhibit protonic conductivity that increases with increasing relative humidity (RH), which is a typical feature for numerous water-mediated proton conductors. The traditional Arrhenius-type plots demonstrate a linear behaviour with a maximum protonic conductivity of 1.6 mS cm-1 at 94 °C and 98 % RH. The results showed that these fully bio-based conductive membranes have potential as eco-friendly alternatives to other PEMs for application in PEFCs.publishe

Natural-based antioxidant extracts as potential mitigators of fruit browning

Fruit enzymatic browning (EB) inhibition continues to be a challenge in the Food Industry. This physiological disorder results mainly from the oxidation of natural phenolic compounds by polyphenoloxidase (PPO) and peroxidase (POX) leading to the formation of brown pigments. EB can be controlled with the application of antioxidants, reducing/inhibiting the activity of these oxidative enzymes. In this study, strawberry tree (leaves and branches) and apple byproduct were the natural-based extracts (NES) selected, as potential tissue browning inhibitors, within a first screening of fifteen natural-based extracts with antioxidant properties. Phenolic profile, total phenolic content and antioxidant activity of the selected extracts were also performed as well as their depletion effect on the oxidative enzyme’s activity and browning inhibiton in fresh-cut pears. Strawberry tree extracts (leaves and branches) revealed higher total phenolic content (207.97 ± 0.01 mg GAE.gNES−1 and 104.07 ± 16.38 mg GAE.gNES−1, respectively), confirmed by the plethora of phenolic compounds identified by LC-ESI-UHR-QqTOF-HRMS and quantified by HPLC. This phytochemical composition was reflected in the low IC50 against PPO and POX obtained. Despite the lower phenolic content (6.76 ± 0.11 mg GAE.gNES−1) and antioxidant activity (IC50 = 45.59 ± 1.34 mg mL−1), apple byproduct extract showed potential in delaying browning. This study highlights the opportunity of byproducts and agricultural wastes extracts as novel anti-browning agents.info:eu-repo/semantics/publishedVersio

Outdoor tourism in the North of Portugal from the perspective of tourist entertainment companies (TEC)

Based on the perception of those responsible for Tourist Entertainment Companies (TEC), the objective of the research is to contribute to the characterization of Outdoor Tourism (OT). Exploratory qualitative research was carried out through 38 in-depth individual interviews. The territory geography, the experiences, and the internal market were considered relevant aspects. In addition, seasonality and climate were identified as significant factors in defining the offer, which can favour the preservation of the environment. Moreover, the risk of the activities was considered low. The interviewees suggested integrating everyone involved in a network to explore the region's potential, as well as to support activities and promote the region. The radical experience, adventure, and getting out of the routine were identified as motivations for the search, as well as contact with nature and the landscape. The research is limited to the North Region of Portugal, and it brings together the most different needs, desires, and expectations for tourism demand. Strengthening OT can favour employability and, consequently, economic and social balance while promotion of the activity can be explored through the customers' previous experience. The evaluations, testimonials, and word of mouth (WOM) being added value that can be optimized through social networks and digital marketing.This project is being carried out within the scope of the TURNOUT Project-Development of Outdoor Tourism of Northern Portugal, POCI-01-0145-ERDF-032289, financed by the European Investment Fund Regional Development (ERDF) through the Operational Program of Competitiveness and Innovation (POCI) and the Foundation for Science and Technology, IP (OE). UNIAG, R&D unit funded by the FCT-Portuguese Foundation for the Development of Science and Technology, Ministry of Science, Technology and Higher Education. “Project Code Reference: UIDB/04752/2020”. This project also thanks to the reviewers of the ICTR21 conference for their improvement inputs to the paper.info:eu-repo/semantics/publishedVersio

Building better biobetters: from fundamentals to industrial application

Biological drugs or biopharmaceuticals off patent open a large market for biosimilars and biobetters, follow-on biologics. Biobetters, in particular, are new drugs designed from existing ones with improved properties such as higher selectivity, stability, half-life and/or lower toxicity/immunogenicity. Glycosylation is one of the most used strategies to improve biological drugs, nonetheless bioconjugation is an additional alternative and refers to the covalent attachment of polymers to biological drugs. Extensive research on novel polymers is underway, nonetheless PEGylation is still the best alternative with the longest clinical track record. Innovative trends based on genetic engineering techniques such as fusion proteins and PASylation are also promising. In this review, all these alternatives wereexplored as well as current market trends, legislation and future perspectives.publishe

Chemical characterization of Sambucus nigra L. flowers aqueous extract and its biological implications

The main goal of this study was to chemically characterize an aqueous S. nigra flower extract and validate it as a bioactive agent. The elderflower aqueous extraction was performed at different temperatures (50, 70 and 90 °C). The extract obtained at 90 °C exhibited the highest phenolic content and antiradical activity. Therefore, this extract was analyzed by GC-MS and HPLC-MS, which allowed the identification of 46 compounds, being quercetin and chlorogenic acid derivatives representative of 86% of the total of phenolic compounds identified in hydrophilic fraction of the aqueous extract. Naringenin (27.2%) was the major compound present in the lipophilic fraction. The antiproliferative effects of the S. nigra extract were evaluated using the colon cancer cell lines RKO, HCT-116, Caco-2 and the extracts antigenotoxic potential was evaluated by the Comet assay in RKO cells. The RKO cells were the most susceptible to S. nigra flower extract (IC50=1250 µg mL1). Moreover, the extract showed antimicrobial activity against Gram-positive bacteria, particularly Staphylococcus aureus and S. epidermidis. These results show that S. nigra-based extracts can be an important dietary source of bioactive phenolic compounds that contribute to health-span improving life quality, demonstrating their potential as nutraceutical, functional foods and/or cosmetic components for therapeutic purposes.This research was funded by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UIDB/04469/2020 unit and for Scientific Employment Stimulus 2017 (CEECIND/01507/2017) provided to Ana M. Sousa.info:eu-repo/semantics/publishedVersio

The use of a geographic information system to increase outdoor tourism

Purpose: Outdoor tourism is a growing segment that still requires data for planning and management. Aimed to present the potential of the Geographic Information System (GIS) to improve outdoor tourism in the North of Portugal. To achieve this purpose, a case study was conducted on developing a Web-GIS in Northern Portugal. Methodology: Four steps were followed to develop the GIS tool with information about outdoor tourism in the North of Portugal. In the first step, municipalities in the North of Portugal sent information about outdoor activities. In the second step, the data were georeferenced and associated. In the last step, a Web-GIS was developed. Findings: The development of tools is an important source of support for tourism. This study identified that a tool such as the GIS could support a destination's decision-making and promotional processes, besides providing detailed information to the tourist and facilitating travel planning. Practical implications: GIS provides effective planning of outdoor tourism, once this tool allows an effective search of the tourist offer and, from the tourist's perspective, relevant suggestions of visits according to their location are presented, facilitating their experience in the region. Originality: Several studies relate to GIS and tourism, but there is still a gap in specific studies on outdoor activities. So, this study presents the importance of GIS to improve outdoor tourism in the North region of Portugal. Understanding this importance is essential in planning and managing tourism, helping further policymaking and marketing strategies.info:eu-repo/semantics/publishedVersio

Extraction and purification of violacein from Yarrowia lipolytica cells using aqueous solutions of surfactants

BACKGROUND: L-Asparaginase (ASNase) is an important biopharmaceutical for the treatment of acute lymphoblastic leukemia (ALL); however, with some restrictions due to its high manufacturing costs. Aqueous biphasic systems (ABS) have been suggested as more economical platforms for the separation/purification of proteins, but a full understanding of the mechanisms behind the ASNase partition is still a major challenge. Polymer/salt-based ABS with different driving-forces (salting-out and hydrophilicity/hydrophobicity effects) were herein applied to control the partition of commercial ASNase. RESULTS: The main results showed the ASNase partition to the salt- or polymer-rich phase depending on the ABS studied, with extraction efficiencies higher than 95%. For systems composed of inorganic salts, the ASNase partition was controlled by the polyethylene glycol (PEG) molecular weight used. Cholinium-salts-based ABS were able to promote a preferential ASNase partition to the polymer-rich phase using PEG-600 and to the salt-rich phase using a more hydrophobic polypropylene glycol (PPG)-400 polymer. It was possible to select the ABS composed of PEG-2000 + potassium phosphate buffer as the most efficient to separate the ASNase from the main contaminant proteins (purification factor = 2.4 ± 0.2), while it was able to maintain the enzyme activity for posterior application as part of a therapeutic. CONCLUSION: Polymer/salt ABS can be used to control the partition of ASNase and adjust its purification yields, demonstrating the ABS potential as more economic platform for the selective recovery of therapeutic enzymes from complex broths.publishe