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- Development and validation method for simultaneous quantification of phenolic compounds in natural extracts and nanosystemsPublication . Silva, Sara Baptista da; Oliveira, Ana; Ferreira, Domingos; Sarmento, Bruno; Pintado, ManuelaIntroductionSage and savoury (Salvia sp. and Satureja montana, respectively) are plants used in traditional medicine. The quality control of their herbal formulations is of paramount concern to guarantee the expected biological activity of their anti-oxidant compounds. Objective To establish a simple and effective high-performance liquid chromatographic (HPLC) method to evaluate simultaneously quercetin and rosmarinic acid, in a pure form, in natural extracts (sage and savoury), and encapsulated into chitosan nanoparticles. Methods Chromatography was performed on an RP C-18-column, in a gradient mode with a mobile phase comprising methanol:formic acid:water 92.5:2.5:5 (v/v) at a flow rate of 0.75mL/min and at wavelength of 280nm. Results The method was specific, linear in the range of 0.05-1mg/mL (R-2=1.00), precise at the intraday and interday levels, accurate (recovery rate 90.50.6%), and robust to changes in equipment conditions. Conclusion The method established was effective for quercetin and rosmarinic acid characterisation in natural extracts and in chitosan nanoparticles, allowing the loading capacity determination, the association efficiency as well as the in vitro release. This study shows a new HPLC method validated to simultaneous quantification of rosmarinic acid and quercetin in natural extracts and chitosan nanoparticles. This method is also able for detection and quantification of other phenolic and flavanoid compounds in natural matrix.
- Natural extracts into chitosan nanocarriers for rosmarinic acid drug deliveryPublication . Silva, Sara Baptista da; Amorim, Manuela; Fonte, Pedro; Madureira, Raquel; Ferreira, Domingos; Pintado, Manuela; Sarmento, BrunoContext: Nanotechnology can be applied to deliver and protect antioxidants in order to control the oxidative stress phenomena in several chronic pathologies. Chitosan (CS) nanoparticles are biodegradable carriers that may protect antioxidants with potent biological activity such as rosmarinic acid (RA) in Salvia officinalis (sage) and Satureja montana (savory) extracts for safe and innovative therapies. Objective: Development and characterization of CS nanoparticles as a stable and protective vehicle to deliver RA for medical applications using natural extracts as sage and savory. Materials and methods: Antioxidant-CS based nanoparticles were prepared by ionic gelation with sodium tripolyphosphate (TPP), at pH 5.8 with a mass ratio of 7:1 (CS:TPP), with a theoretical antioxidant-CS loading of 40-50%. The nanoparticles were then characterized by different methods such as photon correlation spectroscopy, laser Doppler anemometry, scanning electron microscopy (SEM), differential scanning calorimetry (DSC), Fourier-transform infrared (FTIR), high-performance liquid chromatographic (HPLC), association efficiency, and antioxidant activity. Results and discussion: Individual and small sizing nanoparticles, around 300 nm, were obtained. SEM confirmed smooth and spherical nanoparticles after freeze-drying. No chemical interactions were found between antioxidants and CS, after encapsulation, by DSC and FTIR. The association efficiency was 51.2% for RA (with 40% loading) and 96.1 and 98.2% for sage and savory nanoparticles, respectively (both with 50% loading). Antioxidant activity values were higher than 0.0348 eq [Asc. Ac.] g/L/g extract and 0.4251 μmol/eq Trolox/g extract. Conclusion: The extracts under study are promising vehicles for RA drug delivery in CS nanocarriers.
- Chitosan-based nanoparticles for rosmarinic acid ocular delivery-in vitro testsPublication . Silva, Sara; Ferreira, Domingos; Pintado, M. E.; Sarmento, BrunotIn this study, chitosan nanoparticles were used to encapsulate antioxidant rosmarinic acid, Salvia offici-nalis (sage) and Satureja montana (savory) extracts as rosmarinic acid natural vehicles. The nanoparticleswere prepared by ionic gelation using chitosan and sodium tripolyphosphate (TPP) in a mass ratio of7:1, at pH 5.8. Particle size distribution analysis and transmission electron microscopy (TEM) confirmedthe size ranging from 200 to 300 nm, while surface charge of nanoparticles ranged from 20 to 30 mV.Nanoparticles demonstrate to be safe without relevant cytotoxicity against retina pigment epithelium(ARPE-19) and human cornea cell line (HCE-T). The permeability study in HCE monolayer cell line showedan apparent permeability coefficient Pappof 3.41 ± 0.99 × 10−5and 3.24 ± 0.79 × 10−5cm/s for rosmarinicacid loaded chitosan nanoparticles and free in solution, respectively. In ARPE-19 monolayer cell line thePappwas 3.39 ± 0.18 × 10−5and 3.60 ± 0.05 × 10−5cm/s for rosmarinic acid loaded chitosan nanoparti-cles and free in solution, respectively. Considering the mucin interaction method, nanoparticles indicatemucoadhesive proprieties suggesting an increased retention time over the ocular mucosa after instilla-tion. These nanoparticles may be promising drug delivery systems for ocular application in oxidative eyeconditions.