Polymeric Nanoparticles as Oral Delivery Systems for Encapsulation and Release of Polyphenolic Compounds: Impact on Quercetin Antioxidant Activity & Bioaccessibility Hector Pool, David Quintanar, Juan de Dios Figueroa, J. Etelvino H. Bechara, David Julian McClements & Sandra Mendoza Food Biophysics ISSN 1557-1858 Food Biophysics DOI 10.1007/s11483-012-9266-z
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Author's personal copy Food Biophysics DOI 10.1007/s11483-012-9266-z
ORIGINAL ARTICLE
Polymeric Nanoparticles as Oral Delivery Systems for Encapsulation and Release of Polyphenolic Compounds: Impact on Quercetin Antioxidant Activity & Bioaccessibility Hector Pool & David Quintanar & Juan de Dios Figueroa & J. Etelvino H. Bechara & David Julian McClements & Sandra Mendoza Received: 2 April 2012 / Accepted: 8 July 2012 # Springer Science+Business Media, LLC 2012
Abstract A delivery system containing polymeric (Eudragit) nanoparticles has been developed for encapsulation and controlled release of bioactive flavonoids (quercetin). Nanoparticles were fabricated using a solvent displacement method. Particle size, morphology, and charge were measured by light scattering, electron microscopy and ζ-potential. Encapsulation efficiency (EE) and release profiles were determined using electrochemical methods. Molecular interactions within the particle matrix were characterized by X-ray diffraction, differential scanning calorimetry, and infrared spectroscopy. Antioxidant properties of free and encapsulated quercetin were analyzed by TBARS and fluorescence spectroscopy. Bioaccessibility of quercetin was evaluated using an in vitro H. Pool : S. Mendoza (*) Departamento de Investigación y Posgrado en Alimentos, Facultad de Química, Universidad Autónoma de Queretaro, Queretaro, Queretaro 76010, Mexico e-mail:
[email protected] D. Quintanar Laboratorio de Posgrado en Tecnología Farmacéutica, Universidad Nacional Autónoma de México, Cuautitlán Izcalli, Estado de México 54740, Mexico J. d. D. Figueroa Centro de Investigación y Estudios Avanzados del Instituto Politécnico Nacional, unidad Queretaro, Queretaro 76230, Mexico J. E. H. Bechara Laboratorio de Radicais Livres e Estados Excitados en Sistemas Biologicos, Universidade de São Paulo, São Paulo, SP 05508-000, Brazil D. J. McClements Biopolymers and Colloids Research Laboratory, Department of Food Science, University of Massachusetts, Amherst, MA 01003, USA
digestion model. Relatively small (d≈370 nm) anionic polymeric nanoparticles were formed containing quercetin in a non-crystalline form (EE≈67 %). The main interaction between quercetin and Eudragit was hydrogen bonding. Encapsulated quercetin remained stable during 6 months storage and maintained its antioxidant activity. Quercetin bioaccessibility within simulated small intestinal conditions was improved by encapsulation. The knowledge obtained from this study will facilitate the rational design and fabrication of polymeric nanoparticles as oral delivery systems for encapsulation, protection, and release of bioactive compounds. Keywords Quercetin . Nanoparticles . Encapsulation . In vitro digestion . Oxidation . Bioaccessibility Abbreviations Quercetin (QC) Eudragit GIT PVA KBr HCl NaOH KCl DMSO PI SEM EE DPV DSC XRD DRIFT pc
3,3',4',5,7-pentahydroxy flavone Eudragit L30-D55 gastrointestinal tract polyvinil alcohol potassium bromide hydrochloric acid sodium hydroxide potassium chloride dimethyl sulphoxide Polydispersity index Scanning Electron Microscopy drug entrapment efficiency differential pulse voltammetry differential scanning calorimetry X-ray diffraction diffuse reflectance of infrared by fourier transform soybean phosphatidylcholine1
Author's personal copy Food Biophysics
MDA malondialdehyde PBS phosphate buffer C11-BODIPY581/4,4-difluoro-5-(4-phenyl-1,3-butadienyl)591 4-bora-3a, 4a-diaza-s-indaceno-3undecanoic AAPH 2,2′-azobis (2′-methilpropanamidine) dihidrochlorine ROO− peroxy radicals nm nanometers mM millimolar
Introduction Flavonoids are polyphenolic compounds found in many plants and fruits that have been reported to exhibit certain bioactive properties that may be beneficial to human health e.g., antioxidant activity2. Quercetin (3,3′,4′,5,7-pentahydroxy flavone) (Figure 1) is a flavonoid that has been reported to have a particularly highantioxidant activity3,4. Quercetin has alsobeen reported to offer protection against a variety of chronic diseases, such as cardiovascular diseases, inflammation, obesity, bacterial and viral infections, cancer, and other age related diseases2,5–7. The utilization of bioactive compounds (“nutraceuticals”) to formulate functional foods designed to promote human health and wellness has recently received great interest within the food industry8. However, the use of quercetin to produce functional foods has been limited by its poor water solubility, chemical instability under conditions encountered during food processing and storage (e.g., temperature, oxygen, light), and its relatively low bioavailability. In addition, some studies have shown that the antioxidant capacity of flavonoid compounds (such as quercetin) decrease dramatically when they are exposed to acidic or alkaline regions of the gastrointestinal tract (GIT)9. These factors limit the biological activity and potential health benefits of this flavonoid in functional food products10. Consequently, it is necessary to develop effective delivery systems to encapsulate, protect, and release quercetin so that it can be successfully incorporated into foods, and so that
Fig. 1 Chemical structure of quercetin
it maintains its integrity until it is released at the appropriate site of action in the GIT11. The purpose of this study was to determine whether quercetin could be successfully encapsulated, protected, and released by polymer nanoparticles. These nanoparticles were fabricated from a commercial biocompatible polymer (Eudragit L30-D55) that is stable under acidic conditions (pH5.5. It should therefore be possible to design polymeric nanoparticles that encapsulate and protect bioactive compounds within acidic environments (such as some foods and the stomach), and then release them in neutral environments (such as the mouth or small intestine). We therefore investigated the effect of storage pH on the release of quercetin from Eudragit nanoparticles. The in vitro release profiles of quercetin from Eudragit nanoparticles stored at different pH values (2, 4.5 and 7.2) showed that the rate and extent of quercetin release was strongly pH-dependent (Figure 7). At pH 7.2, there was a rapid initial release of quercetin from the nanoparticles during the first four hours, followed by a more gradual increase at longer times, until over 90 % was released. The rapid release of quercetin suggests that the Eudragit nanoparticles started to dissolve at this pH allowing the encapsulated quercetin molecules to escape to the diffusion media. At pH 2, less than 7 % of the quercetin was released from the nanoparticles after 34 h storage, which suggested that the Eudragit nanoparticles remained intact with the quercetin molecules trapped inside them. At pH 4.5, a rapid initial release of about 10 % of quercetin occurred during the first four hours, and then there was only a slight further increase in the amount released at longer times. Presumably, some of the quercetin molecules at the surface of the
Author's personal copy Food Biophysics 100
Antioxidant Capacity of Quercetin A
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Fig. 7 Percentage of quercetin released from Eudragit L30-D55 nanoparticles in Britton-Robinson buffer at pH 7.2 (a), 4.5 (b) and 2 (c). Results are expressed as average±SD of three individual experiments
nanoparticles were released in the initial period, whereas that in the interior of the nanoparticles remained encapsulated. Overall these results suggest that Eudragit remains stable and protects bioactive compounds under acidic pH conditions, but releases them at neutral pH conditions, such as in the small intestine and colon of the human body. Our results are consistent with those reported by other authors using similar systems35,42. The release of quercetin from Eudragit nanoparticles within simulated GIT conditions is discussed later. Storage Stability of Nanoparticles The physicochemical properties of quercetin-free and quercetin-loaded Eudragit nanoparticles were evaluated after storage for 6 months to establish their long-term stability. Freeze dried samples were stored in amber vials placed in desiccators containing drying salts under vacuum. After 6 month storage, there was no significant change (p 80 %) of the encapsulated bioactive component.
Lipid oxidation is one of the most problematic deterioration processes occurring in foods containing unsaturated lipids, which leads to the development of rancidity, off-flavors, and polymerization reactions that reduce the shelf life and nutritive value of food products. Lipid oxidation may also generate undesirable biologically active species that are involved in cardiovascular and inflammatory disease processes43–45. For these reasons, we evaluated the ability of free and encapsulated quercetin to inhibit lipid peroxidation of phospholipid liposomes. The ability of quercetin to inhibit lipid peroxidation of phospholipid liposomes was monitored by measuring the production of malondialdehyde (MDA) for 24 h using the TBARS method (Figure 8). The results were compared with a negative control (buffer solution) and a positive control (buffer solution containing phospholipid liposomes, H2O2 and Fe2+). As expected, there was little change in the amount of MDA formed during storage for the negative control, whereas there was a substantial increase in MDA formation for the positive control. When free or encapsulated quercetin was incorporated into the phospholipid liposome solution 2 h before the oxidation reaction was initiated, the amount of MDA produced during storage was reduced compared to the positive control, with the amount of MDA produced decreasing with increasing quercetin concentration. The effectiveness of the quercetin as an antioxidant depended on whether it was encapsulated or not. At similar quercetin levels, lower amounts of MDA were produced for the encapsulated quercetin than for free quercetin, however, no significant differences (P
