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- Effects of whey peptide extract on the growth of probiotics and gut microbiotaPublication . Yu, Ya-Ju; Amorim, Manuela; Marques, Cláudia; Calhau, Conceição; Pintado, M. E.Whey peptide extract with molecular weight below 1 kDa was investigated in microplate assay, and viable cells, as well as metabolic activity were determined to evaluate augmented growth of probiotic bacteria (Lactobacillus acidophilus and Bifidobacterium animalis). Results illustrated that whey peptide extract 1% (w/v) has the capacity to stimulate the proliferation of both probiotic bacteria tested, further supported by the faster generation of metabolic products. The effect of whey peptide extract on the modulation of gut microbiota was also examined inWistar rats fed either with a standard or a high-fat diet, assessed via 16S ribosomal RNA expression of gut microbiota by quantitative PCR. Relative abundance of Lactobacillus spp., Bifidobacterium spp. and Bacteroidetes was significantly increased by whey peptide extract in rats fed with a standard diet. These results highlight an additional unexploited positive effect of whey peptide extract on gut microbiota modulation.
- Effect of chronic consumption of blackberry extract on high-fat induced obesity in rats and its correlation with metabolic and brain outcomesPublication . Meireles, Manuela; Rodríguez-Alcalá, Luís M; Marques, Cláudia; Norberto, Sónia; Freitas, Joana; Fernandes, Iva; Mateus, Nuno; Gomes, Ana Maria; Faria, Ana; Calhau, ConceiçãoFlavonoids have been presented as potential protectors against metabolic and cognitive dysfunction. However, mechanisms underlying these 'claims' have not been sufficiently explored. To analyse the effect of long-term supplementation with blackberry extract (BE) in the context of a high-fat or a standard diet, Wistar rats were divided into 4 groups (n = 6) fed with a standard or a high-fat diet, with or without BE supplementation at 25 mg per kg body weight per day. A high-fat diet significantly impaired glucose tolerance and increased body weight, caloric ingestion, very-low-density lipoprotein, triglycerides and cholesterol. Furthermore, it was observed that a high-fat diet increased dopamine content in the prefrontal cortex and decreased brain derived neurotrophic factor (BDNF) levels both in the prefrontal cortex and in plasma. BE supplementation only affected some of these aspects. BE slightly improved glucose metabolism and significantly decreased levels of lactate, independent of diet. BE decreased levels of BDNF and also interacted with the dopaminergic system, increasing dopamine turnover in the striatum, and reverting dopamine content induced by a high-fat diet in the prefrontal cortex. This study shows that, despite some particular benefits of anthocyanin supplementation, some long-term effects may not be desirable and further studies are needed to optimize ingestion conditions.
- Fermentation of bioactive solid lipid nanoparticles by human gut microfloraPublication . Madureira, Ana Raquel; Campos, Débora; Gullon, Beatriz; Marques, Cláudia; Rodríguez-Alcalá, Luís M.; Calhau, Conceição; Alonso, Jose Luis; Sarmento, Bruno; Gomes, Ana M.; Pintado, M. E.Solid lipid nanoparticles (SLNs) can be used for oral delivery of phenolic compounds in order to protect them from the harsh conditions of digestion and improve their bioavailability in the intestinal epithelium. Recently, the production and characterization of SLNs loaded with rosmarinic acid (RA) and herbal extracts was performed for future use as functional food ingredients. Diet components have been shown to have a huge impact on gut microbiota viability and metabolic activity. Hence, SLNs loaded with RA, sage and savoury extracts have been evaluated for their effect on intestinal microbiota growth and the metabolic products generated. Fermentations in anaerobic batch cultures using volunteer human faeces were performed during 24 h. Dynamic bacterial population changes were analysed using PCR-real time, as well as the generation of fatty acids and the quantification of phenolic compounds by analytical methods. Solid lipid nanoparticles released phenolic compounds at non-inhibitory bacterial growth concentrations. Released herbal extract phenolic compounds showed a beneficial effect on gut microbiota growth (e.g. bifidogenic effects) and were used as substrates. Acetate, formate, lactate and butyrate were produced in higher concentrations. The released phenolic compounds also induced PUFA and trans fatty acids metabolic activity, the production of saturated fatty acids, as well of potential beneficial conjugated linoleic acid isomers. Solid lipid nanoparticles modulate gut microbiota and metabolic activities.
- In vitro ACE-inhibitory peptide KGYGGVSLPEW facilitates noradrenaline release from sympathetic nerve terminals: relationship with the lack of antihypertensive effect on spontaneous hypertensive ratsPublication . Marques, Cláudia; Amorim, Maria Manuela; Pereira, Joana Odila; Guardão, Luísa; Martins, Maria João; Pintado, Manuela Estevez; Moura, Daniel; Calhau, Conceição; Pinheiro, HélderThis study aimed to validate the antihypertensive activity of the angiotensin-converting enzyme (ACE)-inhibitor whey protein hydrolysate (WPH) obtained through the action of proteolytic enzymes fromCynara Cardunculus. The antihypertensive activity of WPH fractions containing peptides with molecularweight below 3 kDa (Whey < 3 kDa) and 1 kDa (Whey < 1 kDa) along with the antihypertensive activity ofthree potent ACE-inhibitory peptide sequences (DKVGINYW, DAQSAPLRVY and KGYGGVSLPEW), previ-ously identified in WPH, were also investigated. In parallel, the influence of KGYGGVSLPEW (the mostpotent ACE-inhibitory peptide sequence) on AT1receptors (a common pharmacological target of anti-hypertensive therapies beyond ACE), was evaluated. The effect of WPH and fractions (300 mg/kg) andpeptide sequences (5 mg/kg) on systolic, diastolic and mean blood pressure was evaluated by telemetryon Spontaneously Hypertensive Rats (SHR), after single oral administration. Despite their ACE-inhibitoryeffect in vitro, neither WPH, Whey <3 kDa, Whey <1 kDa or peptide sequences exhibited antihyperten-sive activity. In addition, KGYGGVSLPEW was not only devoid of AT1receptor antagonism but, on thecontrary, had a similar effect to that of Ang II by facilitating the noradrenaline release from sympatheticnerve terminals. In vitro ACE blockade does not always correlate with antihypertensive activity and food-derived peptides cannot be classified as antihypertensive agents based exclusively on in vitro assays. Theabsence of an antihypertensive effect may also be a result of the interaction of these compounds withother components of the systems involved in the blood pressure control.
- Safety profile of solid lipid nanoparticles loaded with rosmarinic acid for oral use: in vitro and animal approachesPublication . 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.