Browsing by Author "Ineu, Rafael P."
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- Bioactivity screening of pinhão (Araucaria Angustifolia (Bertol.) Kuntze) seed extracts: the inhibition of cholinesterases and α-amylases, and cytotoxic and anti-inflammatory activitiesPublication . Oliveira, Anielle; Moreira, Thaysa Fernandes Moya; Pepinelli, Ana Luísa; Costa, Luis Gustavo Médice Arabel; Leal, Luana Eloísa; Silva, Tamires Barlati Vieira; Gonçalves, Odinei Hess; Ineu, Rafael P.; Dias, Maria Inês; Barros, Lillian; Abreu, Rui M.V.; Ferreira, Isabel C.F.R.; Bracht, Lívia; Leimann, Fernanda VitóriaThe objective of this work was to determine the potential bioactive properties of extracts from bio-residues of pinhão (Araucaria angustifolia (Bertol.) Kuntze) seeds, namely the α-amylase and cholinesterase inhibition, cytotoxicity, and anti-inflammatory properties. The pinhão extracts evaluated were obtained from cooking water (CW) and as an ethanolic extract from residual pinhão seed shells (PS). Catechin was the major compound found in both extracts. The PS extract presented higher antioxidant levels and the better inhibition of human salivary and porcine pancreatic α-amylases when compared to the CW extract. Also, based on in vivo evaluations, the PS extract did not differ significantly from acarbose when compared to a control group. The most potent inhibitor of cholinesterases was the CW extract. No cytotoxicity toward normal cells was detected, and neither extract showed anti-inflammatory activity. The PS extract presented cytotoxic activity toward non-small-cell lung, cervical, hepatocellular and breast carcinoma cell lines. Overall, the results demonstrated the potential bioactivity of extracts obtained from pinhão bioresidues.
- Chemometric evaluation of enzymatic hydrolysis in the production of fish protein hydrolysates with acetylcholinesterase inhibitory activityPublication . Moreira, Thaysa Fernandes Moya; Pessoa, Luiz Gustavo Antunes; Seixas, Flávio Augusto Vicente; Ineu, Rafael P.; Gonçalves, Odinei Hess; Leimann, Fernanda Vitória; Ribeiro, Ricardo PereiraFish protein hydrolysates (FPH) obtained from industrial processing residues are sources of bioactive peptides. The enzymatic hydrolysis process is essential in obtaining specific bioactivities such as inhibition of the enzyme acetylcholinesterase (AChE). In this study the effect of different hydrolysis conditions on the properties of FPH to inhibit the enzyme acetylcholinesterase. A chemometric evaluation, based on a central composite rotatable design and principal component analysis, was applied to select hydrolysis conditions with best yield, degree of hydrolysis and acetylcholinesterase inhibition. Experimental design results for AChE inhibition were between 10.51 and 40.45% (20, 30 and 50 mg.mL-1 of FPH), and three hydrolysis conditions were selected based on PCA evaluation. The amino acids profile, FTIR and AChE inhibition kinetics were evaluated. Results showed a mixed type of inhibition behavior and, the docking molecular analyzes suggest that the inhibition AChE occurred due to the basic amino acids, mainly by arginine.
- Determination of an optimum extraction region for the recovery of bioactive compounds from olive leaves (Olea europaea L.) using green dynamic pressurized liquid extractionPublication . Silva, Patricia Sabino da; Viell, Franciele Leila Giopato; Ineu, Rafael P.; Bona, Evandro; Dias, Maria Inês; Ferreira, Isabel C.F.R.; Barros, Lillian; Gonçalves, Odinei Hess; Leimann, Fernanda Vitória; Cardozo-Filho, LucioPressurized liquid extraction with water and ethanol was employed to obtain high antioxidant extracts from olive leaves (Olea europaea L.). The influence of solvent ratio (100-50% v/v water/ethanol), temperature (100-200 degrees C), and solvent flow rate (1-5 mL min(-1)) on the extraction process was investigated using a Box-Behnken experimental design. ANOVA was applied to determine the overall goodness of fit of the dependent variables (extraction yield, antioxidant capacity by DPPH, total flavonoids, total iridoid-glycoside, and total phenolic content) in second-order models. For the optimization, these models were used to find the optimal conditions using the Sequential Simplex optimization procedure combined with Derringer and Suich's desirability function to maximize antioxidant capacity, total iridoid-glycoside recovery, and total phenolic content. The optimal region conditions for extraction were determined at a solvent ratio of 53-65% v/v water/ethanol, the extraction temperature of 100-120 degrees C, and a 3-4 mL min(-1) solvent flow rate. Fifteen phenolic compounds were identified in the samples using HPLC-DAD-ESI/MSn, including two phenolic acids (hydroxytyrosol and hydroxytyrosol glucoside), four flavonols (apigenin-6,8-C-dihexoside, quercetin-3-O-rutinoside, luteolin-O-hexoside, and derivatives), and nine iridoid-glycosides (verbascoside, oleuropein, and derivatives). Oleuropein and derivates were identified as the major phenolic compounds in the extract obtained at optimal conditions as a percentage of 89% of the extract composition.
- Encapsulation of clove oil in nanostructured lipid carriers from natural waxes: Preparation, characterization and in vitro evaluation of the cholinesterase enzymesPublication . Meneses, Alessandra Cristina de; Marques, Elisa Balbi Pinto; Leimann, Fernanda Vitória; Gonçalves, Odinei Hess; Ineu, Rafael P.; Araújo, Pedro Henrique Hermes de; Oliveira, Débora de; Sayer, ClaudiaEugenol is a phenolic compound largely found in the clove essential oil that possesses promising biological activity. However, its low water solubility is a major concern and encapsulation is an alternative to improve water affinity. The objective of this work was to produce nanostructured lipid carriers (NLC) by hot homogenization/ultrasound emulsification and to evaluate the effect of free and encapsulated clove oil on the in vitro cholinesterase enzymes modulation using Drosophila melanogaster (DM) tissue. The NLC composed of a natural wax (carnauba or beeswax) and crodamol showed an average diameter between 121 and 367 nm with good dispersion and colloidal stability. The spherical shape and solid character together with the semi-crystalline environment confirm the formation of NLC. DSC analysis indicated polymorphic transition events of the waxes. In vitro tests using DM demonstrated that free clove oil showed a good inhibition of the butyryl and acetylcholinesterase enzymes above a concentration of 10 mM, with IC50 values of 4.3 and 3.5 mM, respectively. The dispersions of the NLC loaded with clove oil showed a decrease in the IC50 enzymes values, indicating the preservation of the clove essential oil and suggesting an increased in the solubility. Results indicate that NLC dispersions have good potential to be used for foods and cosmetic aqueous formulations possessing biological activity.
- Extraction of bioactive compounds from Curcuma longa L. using deep eutectic solvents: in vitro and in vivo biological activitiesPublication . Oliveira, Grazielle; Marques, Caroline; Oliveira, Anielle; Santos, Amanda de Almeida; Amaral, Wanderlei; Ineu, Rafael P.; Leimann, Fernanda Vitória; Peron, Ana Paula; Igarashi-Mafra, Luciana; Mafra, Marcos R.In this work, deep eutectic solvents (DES-based menthol and cholinium chloride) and the ethanol, temperature, and times were selected to extract bioactive compounds from the rhizome, leaves, and flowers Curcuma longa L., using ultrasound-assisted extraction. Analyzes antioxidant, flavonoids, antimicrobial, chelation Fe2+, inhibition of the cholinesterase's enzymes, cytotoxicity, and genotoxicity in Allium cepa cells were performed. The extracts showed results of iron chelation and antibacterial. Curcuma flowers and leaves' extracts inhibited food spoilage bacteria with values above 45%, with substantial iron-chelating activity above 50%. Extracts obtained by DES based on menthol and lactic acid exhibited a high percentage of inhibition of acetyl and butyryl cholinesterase. In contrast, flower extracts obtained by menthol and acetic acid showed low inhibition of cholinesterase enzyme activity. No extract showed cytotoxicity and genotoxicity. Biological activities showed a high potential for the application of these extracts in the food and pharmaceutical industries.
- In vitro and in vivo evaluation of enzymatic and antioxidant activity, cytotoxicity and genotoxicity of curcumin-loaded solid dispersionsPublication . Silva, Igor; Peron, Ana Paula; Leimann, Fernanda Vitória; Bressan, Getúlio Nicola; Krum, Bárbara Nunes; Fachinetto, Roselei; Pinela, José; Calhelha, Ricardo C.; Barreiro, M.F.; Ferreira, Isabel C.F.R.; Gonçalves, Odinei Hess; Ineu, Rafael P.Curcumin, the main bioactive polyphenolic compound in Curcuma longa L. rhizomes has a wide range of bioactive properties. Curcumin presents low solubility in water and thus limited bioavailability, which decreases its applicability. In this study, cytotoxic effects of curcumin solid dispersions (CurSD) were evaluated against tumor (breast adenocarcinoma and lung, cervical and hepatocellular carcinoma) and non-tumor (PLP2) cells, while cytotoxic and genotoxic effects were evaluated in Allium cepa. The effect of the CurSD on the acetylcholinesterase (AChE), butyrylcholinesterase (BChE), glutathione S-transferase (GST), andmonoamine oxidase (MAO A-B) enzymes was determined, as well as its capacity to inhibit the oxidative hemolysis (OxHLIA) and the formation of thiobarbituric acid reactive substances (TBARS). CurSD are constituted by nanoparticles that are readily dispersible in water, and inhibited 24% and 64% of the AChE and BChE activity at 100μM, respectively. GST activity was inhibited at 30μM while MAO-A and B activity were inhibited at 100μM. CurSD showed cytotoxicity against all the tested tumor cell lines without toxic effects for non-tumor cells. No cytotoxic and genotoxic potential was detected with the Allium cepa test.CurSD maintained the characteristics of free curcumin on the in vitro modulation of important enzymes without appreciable toxicity.
- Influence of nanoencapsulated lutein on acetylcholinesterase activity: In vitro determination, kinetic parameters, and in silico docking simulationsPublication . Miranda, Cristiane Grella; Santos, Priscila Dayane Freitas dos; Silva, Jéssica Thaís do Prado; Leimann, Fernanda Vitória; Borges, Bianca Ferreira; Abreu, Rui M.V.; Ineu, Rafael P.; Gonçalves, Odinei HessLutein is a bioactive found in dark leafy vegetables that may be used as a nutraceutical agent in foodstuff and an inhibitor of key enzymes of the human body such as those involved in the cholinergic system. However, its high hydrophobicity leads to low bioavailability and must be overcome if lutein is to be added in foods. The objective of this study was to evaluate the influence of nanoencapsulated lutein in the activity of the acetylcholinesterase enzyme. The in vitro study was carried out using water in order to evaluate the impact of encapsulation on the hydrophilicity of lutein. In vitro assays showed that lutein, both free and nanoencapsulated, presented a mixedtype inhibition behavior, and encapsulated lutein was able to inhibit acetylcholinesterase activity even in an aqueous medium. Inhibition was also showed by the in silico docking results which show that lutein interacted with the pocket region of the enzyme.
- Nanodispersions of beta-carotene: effects on antioxidant enzymes and cytotoxic propertiesPublication . Rocha, Felipe; Sugahara, Letícia Yumi; Leimann, Fernanda Vitória; Oliveira, Sara Marchesan de; Brum, Evelyne da Silva; Calhelha, Ricardo C.; Barreiro, M.F.; Ferreira, Isabel C.F.R.; Ineu, Rafael P.; Gonçalves, Odinei HessBeta-carotene is a carotenoid precursor of vitamin A, known for its biological activities. Due to its high hydrophobicity, nanonization processes, i.e. the transformation into nanoparticles, can improve its water affinity, and therefore the activity in aqueous systems. The objective of this study was to produce beta-carotene nanoparticles by the solid dispersion method and to evaluate their effects on the activity of glutathione-S-transferase and acetylcholinesterase enzymes using Drosophila melanogaster (DM) homogenate, the superoxide dismutase- and catalase-like activities under in vitro conditions, and their cytotoxic properties against tumor and non-tumor cells. The formed nanometric beta-carotene particles resulted in stable colloids, readily dispersed in water, able to modulate acetylcholinesterase (AChE) activity, and presenting high potential to control the cholinergic system. Beta-carotene nanoparticles, at concentrations much lower than the pure pristine beta-carotene, presented in vitro mimetic activity to superoxide dismutase and altered glutathione-S-transferase activity in DM tissue. The content of hydrogen peroxide was neither affected by the nanoparticles (in aqueous solution) nor by pristine beta-carotene (in DMSO). In the cytotoxic assays, beta-carotene nanoparticles dispersed in water showed activity against four different tumor cell lines. Overall, beta-carotene nanoparticles presented significant bioactivity in aqueous medium surpassing their high hydrophobicity constraint.
- The nanoencapsulation of curcuminoids extracted from: Curcuma longa L. and an evaluation of their cytotoxic, enzymatic, antioxidant and anti-inflammatory activitiesPublication . Santos, Priscila Dayane Freitas dos; Francisco, Cristhian Rafael Lopes; Coqueiro, Aline; Leimann, Fernanda Vitória; Pinela, José; Calhelha, Ricardo C.; Ineu, Rafael P.; Ferreira, Isabel C.F.R.; Bona, Evandro; Gonçalves, Odinei HessCurcumin, bisdemethoxycurcumin and demethoxycurcumin are the main curcuminoids present in Curcuma longa L. and are known for their bioactivity. However, their low water solubility results in poor bioavailability and therapeutic efficacy. This work aimed to investigate the in vitro modulation capacity on the enzymes acetylcholinesterase (AChE) and glutathione S-transferase (GST), as well as the in vitro antioxidant (OxHLIA and TBARS) and anti-inflammatory activities (RAW 264.7 test) of nanoencapsulated curcuminoids. Cytotoxicity on tumor and non-tumor cell lines was also investigated. Curcuminoid nanoparticles significantly inhibited the in vitro activity of AChE (12% inhibition at 50 μM) and GST (30% inhibition at 5 μM). They presented antioxidant activity and toxic effects against breast adenocarcinoma, lung, cervical and hepatocellular carcinoma cells when dispersed in water. Encapsulated curcuminoids exhibited bioactive properties in aqueous medium (no hydrophobic solvent added), exerting antioxidant and cytotoxic effects and acting on the cholinergic and endogenous antioxidant systems.
- Natural food preservatives: application of rosemary, basil, and sage in yogurts as a viable alternative to artificial ones, using sustainable, low cost and efficient processPublication . Ueda, Jonata Massao; Pedrosa, Mariana C.; Fernandes, Filipa Alexandra; Calhelha, Ricardo C.; Melgar Castañeda, Bruno; Rodrigues, Paula; Dias, Maria Inês; Pinela, José; Ivanov, Marija; Soković, Marina; Ineu, Rafael P.; Carocho, Márcio; Ferreira, Isabel C.F.R.; Heleno, Sandrina A.; Barros, LillianFoods are susceptible to various forms of degradation, either by oxygen present in the atmosphere, microbiological factors, enzymatic degradation and others, being essential to study new ways of preserving food. Due to the need of creating “stock” and preserve food in the pre-industrial era, consumers were essentially concerned on obtaining food as quickly as possible, opting for “industrialized” food. Nowadays, the demand for non-processed foods or with a minimum of artificial additives is increasing due to an increased awareness of consumers towards healthier diets, studies related to health risks and public opinions generated in the media [1,2]. Plants are known to be rich sources of bioactive compounds, since these are considered a defense mechanism against external agents such as microorganisms, oxidizing agents and ultraviolet radiation, and for these reasons their extracts are investigated as promising sources of natural additives for the food industry [3]. Thus, the main objective of this work was the exploitation of rosemary (Rosmarinus officinalis L.), basil (Ocimum basilicum L.) and sage (Salvia officinalis L.) as sources of preservative molecules, for incorporation in yogurts. This included: i) the optimization of the extraction of bioactive molecules through ultrasound assisted extraction (UAE), a sustainable and lowcost extraction technology, using green solvents (water and ethanol); ii) chemical characterization of the extracts obtained by HPLC-DAD-ESI/MS techniques; iii) evaluation of the bioactivities of the extracts (antioxidant, antimicrobial and cytotoxic activities); iv) incorporation of the most promising extracts into the yogurts, and v) the evaluation of the stability of the ingredients over the shelf-life through physical parameters (texture, color and pH); microbiological analysis [4]; nutritional value [5] and monitoring the molecules with preservative capacity. All the formulations were compared with control samples (without additives and with potassium sorbate, a common artificial additive used in yogurts). According to the obtained results, for the extraction optimization, it was found that the percentage of solvent is the most relevant factor for obtaining an extract rich in rosmarinic acid, followed by the extraction time and ultrasonic power. For the antioxidant and antimicrobial activity, rosemary extract showed the best result, followed by sage and basil. Moreover, none of the three plant extracts showed hepatotoxicity, at the maximum concentration tested. Finally, the plant extracts did not show changes in the physico-chemical and nutritional characteristics of the yogurts and the incorporation of the preservative extracts did not affect the normal lactic bacteria growth, which are fundamental for the yogurt manufacturing process. Moreover, the extracts were obtained through sustainable, green and low-cost process, providing the industry with safer and economically viable alternatives.