Débora A. Campos

Optimization of the production of solid Witepsol nanoparticles loaded with rosmarinic acid.

During the last decade there has been a growing interest in the formulation of new food and nutraceutical products containing compounds with antioxidant activity. Unfortunately, due to their structure, certain compounds such as polyphenols, in particular rosmarinic acid (RA) are not stable and may interact easily with matrices in which they are incorporated. To overcome such limitations, the formulation of loaded polyphenols nanoparticles can offer an efficient solution to protect such compounds. Based on this rationale, the aim of this study was to prepare solid lipid nanoparticles (SLNs) loaded with RA using a hot melt ultrasonication method, where Witepsol H15 was used as lipid and Polysorbate 80 (Tween 80) as surfactant, following a 3(2) fractional factorial design, resulting in the use of 3 different percentages of surfactant (viz. 1, 2 and 3%, v/v) and lipid (0.5, 1.0 and 1.5%, w/v). The stability of the nanoparticles systems were tested during 28 d in aqueous solution stored at refrigeration temperature (ca. 5 °C), tracking the mean particle size of different formulations by photon correlation spectroscopy. To confirm RA entrapment, thermal analyses of the nanoparticles by DSC and FTIR were performed. The association efficiencies percentages (AE%) were determined using HPLC to quantitatively assess the RA in supernatants. Results showed that Witepsol H15 produced nanoparticles with initial mean diameters between 270 and 1000 nm, yet over time, a slight increase occurred, but without occurrence of aggregation. The AE% showed a high percentage of encapsulation (ca. 99%), which reveals low polyphenol releases from SLNs throughout storage time. In general, results showed a successful production of SLNs with properties that can be used to food applications.

Characterization of solid lipid nanoparticles produced with carnauba wax for rosmarinic acid oral delivery

In the last decade, research studies have increased on the development of delivery systems for polyphenols, for protection, improvement of stability and increase of their bioavailability. Rosmarinic acid is a polyphenol with described bioactivities, such as antioxidant, anti-mutagenic, anti-bacterial and anti-viral capabilities. Thus, the aim of this research work was to produce stable solid lipid nanoparticles (SLN) using carnauba wax as lipidic matrix, for delivery of rosmarinic acid, to be further incorporated into food matrices. Hence, different concentrations of wax (0.5, 1 and 1.5%, w/v) and percentages of surfactant (1, 2 and 3%, v/v) were tested. Physical properties, surface morphology and association efficiencies were studied at time of production and after 28 day at refrigerated storage. Thermal properties and the nature of the chemical interactions between the lipids waxes and rosmarinic acid were also evaluated. The particles showed range size between 35–927 nm and zeta potentials of ca. −38 to 40, showing high stability, with no risk of aggregation due to electric repulsion of SLN. High association efficiencies % (ca. 99%) were obtained. FTIR analyses proved the association of rosmarinic acid and lipidic matrix. The low lipid and high surfactant concentrations leads to small SLN. The surfactant, polysorbate 80 decreases the interfacial tension in the SLN surfaces, preventing aggregation, leading to the development of small particles. These properties were maintained throughout the 28 day of refrigerated storage, and no rosmarinic acid was released by the particles during refrigeration, indicating good compatibility between rosmarinic acid and the waxy core of SLN. The optimum range values to obtain the desirable features for incorporation in a functional food suggest formulations containing 1.0 and 1.5% (w/v) of lipid and 2% (v/v) of surfactant.

Study of antimicrobial activity and atomic force microscopy imaging of the action mechanism of cashew tree gum.

The aim of this work was to evaluate the antimicrobial potential of two grades of cashew tree gum (crude and purified) against eight microorganisms and to analyze the mechanism of cashew tree gum antimicrobial action via atomic force microscopy (AFM) imaging. The results indicated strong antimicrobial properties of pure cashew tree gum against all tested microorganisms, except for Candida albicans and Lactobacillus acidophilus. On the other hand crude cashew gum showed antimicrobial activity only against Gram-positive bacteria (MRSA, MSSA, Listeria innocua and Enterococcus faecium). Atomic force microscopy imaging showed that pure cashew tree gum lead to bacterial cell collapse. In conclusion cashew tree gum presented relevant antimicrobial activity against most of the studied bacteria, and the purification of the cashew gum affected its antimicrobial spectrum.

Therapeutic and nutraceutical potential of rosmarinic acid—Cytoprotective properties and pharmacokinetic profile

ABSTRACT Rosmarinic acid (RA) is a natural polyphenolic antioxidant derived from many common herbal plants. This compound displays several important biological properties, including anti-inflammatory, antiviral, antibacterial, antidepressant, anticarcionogenic, and chemopreventive properties. The importance of its activities and its possible application in processed foods as a natural antioxidant has reached a new interest levels in recent years. The health effects of this polyphenol depend greatly on both its intakes and bioavailability. This review focuses on the importance of RA as a dietary supplement, and summarizes its pharmacokinetics and metabolism, including the factors that limit its oral bioavailability which leads to a lower therapeutic action. Further experimental investigations are needed to optimize and enhance the oral bioavailability of this natural compound which consequently will help increasing therapeutic efficacy of RA in vivo.

Solid lipid nanoparticles as oral delivery systems of phenolic compounds: Overcoming pharmacokinetic limitations for nutraceutical applications

ABSTRACT Drug delivery systems, accompanied by nanoparticle technology, have recently emerged as prominent solutions to improve the pharmacokinetic properties, namely bioavailability, of therapeutic and nutraceutical agents. Solid lipid nanoparticles (SLNs) have received much attention from researchers due to their potential to protect or improve drug properties. SLNs have been reported to be an alternative system to traditional carriers, such as emulsions, liposomes, and polymeric nanoparticles. Phenolic compounds are widespread in plant-derived foodstuffs and therefore abundant in our diet. Over the last decades, phenolic compounds have received considerable attention due to several health promoting properties, mostly related to their antioxidant activity, which can have important implications for health. However, most of these compounds have been associated with poor bioavailability being poorly absorbed, rapidly metabolized and eliminated, which compromises its biological and pharmacological benefits. This paper provides a systematic review of the use of SLNs as oral delivery systems of phenolic compounds, in order to overcome pharmacokinetic limitations of these compounds and improved nutraceutical potential. In vitro studies, as well as works describing topical and oral treatments will be revisited and discussed. The classification, synthesis, and clinical application of these nanomaterials will be also considered in this review article.

Chitosan mouthwash: Toxicity and in vivo validation

A previous study showed that a chitosan mouthwash would be a valid alternative to current mouthwashes as it demonstrated, in vitro, significantly higher antibiofilm activity than two commercial mouthwashes. As such, the aim of this work was to verify the safety of the developed product and to validate, in vivo, the biological activity ascertained in vitro. Chitosan mouthwash safety was evaluated through Ames, MTT and V79 chromosomal aberration assay while antimicrobial activity was evaluated through in vivo assays. The results showed that the chitosan mouthwash was safe, presenting lower cytotoxicity than a commercial mouthwash, and that it effectively reduced viable counts of Streptococcus spp. and Enterococcus spp. by ca. 5.5 log of CFU. Furthermore, in direct comparison with a commercial mouthwash the chitosan mouthwash possessed significantly higher antimicrobial activity. The conjunction of these results proves that the chitosan mouthwash is a safe, effective, natural alternative to the existent chemical mouthwashes.

Insights into the protective role of solid lipid nanoparticles on rosmarinic acid bioactivity during exposure to simulated gastrointestinal conditions.

The evaluation of the digestion effects on bioactive solid lipid nanoparticles (SLN) was performed. For this purpose, witepsol and carnauba SLN loaded with rosmarinic acid (RA) were exposed to the simulated gastrointestinal tract (GIT) conditions prevailing in stomach and small intestine. The simulation of intestinal epithelium was made with a dialysis bag and intestinal cell culture lines. Changes on SLN physical properties, RA release and absorption profiles were followed at each step. Combination of digestion pH and enzymes showed a significant effect upon SLN physical properties. Zeta potential values increased at stomach conditions and decreased at small intestine simulation. Also, at intestine, SLN increased their sizes and released 40-60% of RA, maintaining its initial antioxidant activity values. Sustained release of 40% of RA from SLN was also observed in dialysis tube. At CaCo-2 cell line, both types of SLN showed similar absorbed RA % (ca. 30%). Nevertheless, in CaCo-2/HT29x mix cell lines, for carnauba SLN a lower adsorption RA % was observed than for witepsol SLN. Solid lipid nanoparticles protected RA bioactivity (in terms of antioxidant activity) until reaching the intestine. A controlled release of RA from SLN was achieved and a significant absorption was observed at intestinal cells. Overall, SLN produced with witepsol showed a higher stability than carnauba SLN.

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

ABSTRACT Solid lipid nanoparticles (SLN) can be used as vehicles for phenolic compounds rich extracts. In the present work 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 stability when exposed to simulated gastrointestinal tract (GIT) conditions were also evaluated. Different phenolic compounds from sage 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.

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

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.