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Technological stability of solid lipid nanoparticles loaded with phenolic compounds: drying process and stability along storage

Publication . Campos, Débora A.; Madureira, Ana Raquel; Sarmento, Bruno; Pintado, Maria Manuela; Gomes, Ana Maria

Solid lipid nanoparticles (SLNs) have been widely studied and tested as vehicles for natural compounds. Recently, Witepsol and Carnauba SLNs were shown to be effective systems for the entrapment of rosmarinic acid (RA) and herbal extracts. In the present work, the improvement of stability and bioactivity of these systems was studied. Thus, the freeze-drying of SLNs produced with Witepsol and Carnauba waxes loaded with RA and herbal extracts (sage and savory) were tested. The use of three different cryoprotectants (glucose, mannitol and trehalose) at two different concentrations (5 and 10%, w/v) were evaluated. Furthermore, the prepared SLNs were stored under different conditions (atmosphere, temperature, absence or presence of light) and in different packaging materials, over 365 days. The effect on the SLNs physical stability and bioactivity was assessed. The most suitable cryoprotectant was mannitol at 10% (w/v) for all formulations tested. The solid state of SLNs, with storage at room temperature, in glass flasks, protected from light and under N2 controlled atmosphere were the best storage conditions in which the SLNs bioactivity was maintained during 365 days.

2017Journal article

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Study of the interactions between rosmarinic acid and bovine milk whey protein α-Lactalbumin, β-Lactoglobulin and Lactoferrin

Publication . Ferraro, Vincenza; Madureira, Ana Raquel; Sarmento, Bruno; Gomes, Ana; Pintado, Manuela E.

Incorporation of polyphenols in dairy food is being considered as way to develop oral nutraceutical formulation due to their antioxidant and anti-inflammatory activities. However, polyphenols can interact with proteins, which gives rise to a significant loss of their biological properties. The objective of this research was to study the interactions of rosmarinic acid (RA) and bovine milk whey protein (i.e. α-Lactalbumin, β-Lactoglobulin and Lactoferrin). Radical quenching assay (ABTS), optical density, liquid chromatography (HPLC and FPLC), dynamic light scattering (DLS) and zeta-potential, Fourier transform infrared spectroscopy (FTIR), and differential scanning calorimetry (DSC) were used for the screening of the interactions at 0, 3, and 24 h of storage at 4 °C. Interactions were assessed both at neutral pH of the complexes inwater and acidic pH 3 and 4.5. Results show the occurrence of non-covalent interactions between RA and whey proteins, such as hydrophobic, hydrogen bonding, and dipole–dipole type. Radical quenching activity of RA decreased in the presence of milk proteins yet at the initial time especially in the case of α-Lactalbumin,meaning that the amount of free polyphenol diminished. Complex dimension was different depending on pH, and on primary and secondary structures of proteins. Interactions showed to be favored at the lowest pH, and reversible in all cases; nonetheless, complex RA–proteins were more stable than proteins alone.

2015Journal article

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Current state on the development of nanoparticles for use against bacterial gastrointestinal pathogens: focus on chitosan nanoparticles loaded with phenolic compounds

Publication . Madureira, Ana Raquel; Pereira, Adriana; Pintado, Manuela

Gastrointestinal diseases have a huge impact especially in third world countries, making it urgent to seek new effective antimicrobial therapies. Thus, the development of nanoparticles (NPs) with bioactive compounds having antimicrobial activity has been the target of research over the past years. The development of antimicrobial drug NPs may be promising to overcome the problems associated with antibiotic resistance caused by many pathogenic bacteria. Moreover, the NPs administration of antimicrobial agents has advantages associated therewith, as use of low cost materials, contribution to the improvement of the therapeutic index and a controlled release drug by increasing the pharmacokinetics. These systems can be used to specific strains of bacteria, and to release interesting antimicrobial compounds. The phenolic compounds (PC) are a class of such bioactive compounds for which their antimicrobial activity was already tested on the production of NPs. Polymeric or lipidic NPs systems have been investigated to deliver these compounds. Chitosan is a polymer widely known for their properties, especially the antimicrobial activity and its ability to adhere to intestinal epithelium. This review article aims to evaluate and discuss recent developments in PC new delivery systems with antimicrobial activity against gastrointestinal pathogens, their production processes, activities, focusing on NPs produced using chitosan as the main structural and functional material.

2015Journal article

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Safety profile of solid lipid nanoparticles loaded with rosmarinic acid for oral use: in vitro and animal approaches

Publication . Reis, Flávio; Madureira, Ana Raquel; Nunes, Sara; Campos, Débora; Fernandes, João; Marques, Cláudia; Zuzarte, Monica; Gullón, Beatriz; Rodríguez-Alcalá, Luis M.; Calhau, Conceição; Sarmento, Bruno; Gomes, Ana M.; Pintado, M. E.

Rosmarinic acid (RA) possesses several protective bioactivities that have attracted increasing interest by nutraceutical/pharmaceutical industries. Considering the reduced bioavailability after oral use, effective (and safe) delivery systems are crucial to protect RA from gastrointestinal degradation. This study aims to characterize the safety profile of solid lipid nanoparticles produced with Witepsol and Carnauba waxes and loaded with RA, using in vitro and in vivo approaches, focused on genotoxicity and cytotoxicity assays, redox status markers, hematological and biochemical profile, liver and kidney function, gut bacterial microbiota, and fecal fatty acids composition. Free RA and sage extract, empty nanoparticles, or nanoparticles loaded with RA or sage extract (0.15 and 1.5 mg/mL) were evaluated for cell (lymphocytes) viability, necrosis and apoptosis, and antioxidant/prooxidant effects upon DNA. Wistar rats were orally treated for 14 days with vehicle (control) and with Witepsol or Carnauba nanoparticles loaded with RA at 1 and 10 mg/kg body weight/d. Blood, urine, feces, and several tissues were collected for analysis. Free and loaded RA, at 0.15 mg/mL, presented a safe profile, while genotoxic potential was found for the higher dose (1.5 mg/mL), mainly by necrosis. Our data suggest that both types of nanoparticles are safe when loaded with moderate concentrations of RA, without in vitro genotoxicity and cytotoxicity and with an in vivo safety profile in rats orally treated, thus opening new avenues for use in nutraceutical applications.

2016Journal article

Open access

Stability of bioactive solid lipid nanoparticles loaded with herbal extracts when exposed to simulated gastrointestinal tract conditions

Publication . Campos, Débora A.; Madureira, Ana Raquel; Sarmento, Bruno; Gomes, Ana Maria; Pintado, Maria Manuela

Solid lipid nanoparticles (SLN) can be used as vehicles for phenolic compounds rich extracts. In the presentwork two types of waxes — witepsol and carnauba were tested for the first time in the production of solid lipid nanoparticles (WSLN and CSLN, respectively) loaded with sage and savoury extracts. Physical characterization and association efficiencies calculation were performed. Discrimination of loaded phenolic compounds from each extract was made using HPLC assays. Antioxidant activities of SLN were characterized using two different methods— ABTS and ORAC. Finally, the phenolic compound release profile from SLN and stabilitywhen exposed to simulated gastrointestinal tract (GIT) conditionswere also evaluated. Different phenolic compounds fromsage and savoury extracts were entrapped in SLN. The highest antioxidant activity was obtained for the SLN loaded with savoury extract. Stomach simulated condition provokes a partial release of rosmarinic acid from SLN, whereas at small intestine simulation step, all SLN showed a release of ca. 100%. Witepsol SLN were the ones that best maintained their physical integrity during digestion, showing to be the most stable vehicles for sage and savoury extracts. These SLN show to be suitable for the production of food functional ingredients bearing antioxidant activity.

2015Journal article

Restricted access