Enhanced Delivery of Quercetin by Encapsulation in Poloxamers by

Article pubs.acs.org/IECR

Enhanced Delivery of Quercetin by Encapsulation in Poloxamers by Supercritical Antisolvent Process Marta Fraile, Rafaella Buratto, Beatriz Gómez, Á ngel Martín,* and María José Cocero High Pressure Processes Group, Department of Chemical Engineering and Environmental Technology, Facultad de Ciencias, University of Valladolid, Prado de la Magdalena s/n, 47011 Valladolid, Spain ABSTRACT: The promising applications of quercetin as a functional additive in food and pharmaceutical products are hindered by the low bioavailability of this compound. The formulation of quercetin by encapsulation in a surfactant polymer capable of forming micelles in aqueous media can help improve the dissolution of quercetin in water and thus facilitate the assimilation of this compound by organisms. In this work, quercetin was encapsulated in Pluronic F127 poloxamers by the supercritical antisolvent (SAS) technique. The structure and morphology of the product were characterized by SEM, DSC, XRD, and FTIR spectroscopy. The results indicated that, through SAS processing, a significant reduction in particle size was achieved. With appropriate polymer/active compound mass ratios, quercetin was homogeneously dispersed in an amorphous polymer matrix, and no segregated crystalline particles of quercetin were observed. These structural and morphological variations enabled an improved dissolution behavior of quercetin in simulated gastric and intestinal fluids.

1. INTRODUCTION Quercetin (C15H10O7, molecular weight 302.2 g/mol) is a flavonoid that can be found in many fruits and vegetables such as apples, grapes, strawberries, onions, broccoli, and cabbage, as well as in different drinks such as black tea, red wine, and beer. In addition to its strong antioxidant properties, quercetin shows other important biological activities, including antibacterial, anti-inflammatory, and antihistaminic effects. In the human diet, some benefits of moderate ingestion of quercetin for the treatment of different chronic diseases have been claimed.1 These applications of quercetin are limited by the low bioavailability of this compound. First, quercetin is poorly soluble in aqueous media such as gastrointestinal fluids, with solubilities in water ranging from 2 ppm at 25 °C to 60 ppm at 100 °C.2 Moreover, most ingested quercetin is degraded by gut flora, and only small amounts are absorbed, usually in the form of derived compounds such as glycosides and sulfates.3,4 Therefore, there is considerable interest in the development of formulations of quercetin capable of improving its water solubility, as well as protecting the active groups of the molecule from degradation, that would thus be suitable for increasing the bioavailability and biological activity of this compound. Different formulations of quercetin are described in the literature. Several authors have studied formulations of quercetin with different types of cyclodextrins. The formation of inclusion complexes by the incorporation of quercetin in the inner cavity of these carriers materials was confirmed,5 and certain enhancements in water solubility and antioxidant activity were observed.6,7 The encapsulation or incorporation of quercetin inside a shell of Eudragit E and poly(vinyl alcohol) carriers was studied by Wu et al.,8 who described the formation of solid solutions of carrier and active compound through hydrogen bonding and observed that the product was converted into an amorphous state, conditions that were found to be favorable for increased bioavailability. Li et al.9 described the formation of quercetin-loaded solid lipid © 2014 American Chemical Society

nanoparticles by an emulsification and low-temperature solidification method, observing an increase in the absorption of the active compound by this formulation. Pluronic poloxamers are another class of interesting carrier materials for the enhancement of the absorption of waterinsoluble compounds because of their capacity to form micelles in aqueous environments, hosting hydrophobic compounds.10 Different authors have described the formation of quercetinloaded Pluronic micelles by thin-film hydration methods11 and shown that the resulting micelles enhanced the solubilization of the active compound.12 Ghanem et al. described the encapsulation of quercetin in Pluronic F127 through spraydrying.13 Different methods of producing micellar formulations of poorly water-soluble active compounds in polymeric carriers with surfactant properties such as Pluronic have been described. The conventional technique involves the formation of an oil-inwater emulsion, stabilized by the polymeric carrier, followed by the removal of the organic solvent from the emulsion by evaporation or dialysis.14 This method is hampered by long processing times and harsh operating conditions that result in low encapsulation efficiencies, typically below 30%. Supercritical fluid technologies offer several advantages for the processing of natural compounds. Different techniques based on supercritical carbon dioxide (sc-CO2) enable the processing of sensitive materials at moderate temperatures and in an inert environment, thus avoiding the contamination or degradation of the compounds. Furthermore, such techniques allow the eliminatinon or at least reduction of the use of toxic organic solvents. Because of these advantages, several methods for the precipitation and encapsulation of active compounds Received: Revised: Accepted: Published: 4318

January 9, 2014 February 21, 2014 February 26, 2014 February 26, 2014 dx.doi.org/10.1021/ie5001136 | Ind. Eng. Chem. Res. 2014, 53, 4318−4327

Industrial & Engineering Chemistry Research

Article

Figure 1. Schematic representation of an SAS apparatus.

based on sc-CO2 as the solvent, antisolvent, or solute have been developed. In particular, the supercritical antisolvent (SAS) process is based on the high or complete miscibility of most volatile organic solvents with sc-CO2 at moderate pressures (usually, 8−15 MPa) and the low solubility of many highmolecular-weight solids in CO2 under these conditions. Thus, by mixing an organic solution of the active compound with scCO2, a gas-expanded solution can be formed, producing a very fast and homogeneous precipitation of the compound because of the antisolvent effect of CO2.15−17 By manipulation of the SAS process conditions (pressure, temperature, organic/CO2 ratio, solution concentration, solution atomization, and so on), precise control over the particle size and morphology, as well as the crystalline structure of the particles, can be achieved.16 SAS processing can also be used for the coprecipitation or simultaneous precipitation of an active compound with a carrier material.17 Methods for the production of micellar formulations based on supercritical fluids have also been proposed. Fraile et al.18 developed an emulsion-template process in which the solvent evaporation or displacement process of the conventional methods was substituted by an extraction with supercritical carbon dioxide. Active compounds can also be encapsulated in polymeric micelles by forming a micellar solution in a supercritical fluid that is transformed into solid particles with a micellar structure through the removal of the supercritical solvent by means of a sudden depressurization.19 Tyrrell et al.20 studied the formation of micelles of poly(ethylene glycol)−poly(ε-caprolactone) block cooplymers in supercritical trifluoromethane. Their results showed that polymer micelles were formed in the supercritical fluid at pressures above the cloud point of the polymer in the supercritical fluid whereas, when the pressure was further increased above a critical micelle pressure, a random molecular solution was formed. Furthermore, a powder was produced by removing the supercritical solvent by vaporization induced by sudden depressurization, and it was shown that rehydration of the powder reconstituted the micelles, indicating that the

micellar structure was preserved during the formation of the powder. In a subsequent work, Tyrell et al.21 demonstrated that polymeric micelles formed by this method could be used to encapsulate paclitaxel, a poorly water-soluble cancer therapy drug, achieving encapsulation efficiencies of 87%, significantly higher than the efficiencies of up to 28% obtained by conventional solvent evaporation methods. By testing other active compounds with different solubilities in the supercritical fluid, these authors found that, for successful encapsulation, the precipitation of the active compound along the decompression path should take place at pressures in the range of the micellar solution region. Several authors have studied the processing of quercetin by supercritical technologies. Chafer et al.22 measured the solubility of quercetin in carbon dioxide with ethanol as the cosolvent. They found that the solubility of quercetin in pure carbon dioxide was very low, below 10−6 (molar fraction) even at pressures above 40 MPa, thus demonstrating that supercritical carbon dioxide can act as an effective antisolvent for quercetin. Indeed, several authors have studied the supercritical CO2 extraction of quercetin from natural matrixes, observing that high concentrations of ethanol as the cosolvent and pressures above 40 MPa were required for an efficient extraction.23 Different authors have studied the micronization or reduction of the particle size of pure quercetin to the micrometer range by SAS precipitation. Liu et al.24 studied the precipitation of pure quercetin by the SAS technique, obtaining microparticles of 1−6 μm. Santos and Meireles25 also studied the SAS precipitation of pure quercetin, obtaining needlelike crystals of less than 2 μm. Alessi et al.26 studied the precipitation of pure quercetin by SAS techniques using different organic solvents and employed a thermodynamic model based on the Peng−Robinson equation of state to select the optimum process conditions, observing an increase in the dissolution rate in simulated gastric and intestinal fluids that they attributed to the reduction of the crystallite size by SAS processing. 4319

dx.doi.org/10.1021/ie5001136 | Ind. Eng. Chem. Res. 2014, 53, 4318−4327


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depressurized, and the particles were collected from the filter and stored for further analysis. 2.3. Product Characterization. Particle morphology was analyzed by scanning electron microscopy (SEM) using a JEOL JSM 820 instrument (20 kV, 23-mm working distance). Before analysis, samples were covered with a 50-nm layer of gold using a Balzers SCD004 sputter coater under an argon atmosphere. Differential scanning calorimetry (DSC) profiles were obtained using a Mettler Toledo DSC 822e differential scanning calorimeter in the temperature range of 30−350 °C. Fourier transform infrared (FTIR) spectroscopy was performed using a Bruker ALPHA FTIR spectrometer with a platinum attenuated-total-reflection (ATR) single-diffraction sampling module. X-ray diffraction (XRD) measurements were carried out using a Bruker D8 Discover A25 instrument with a copper ceramic tube of 2.2 kW and a 3 kW generator. The amount of quercetin loaded inside the particles was determined by spectrophotometric analysis at 370 nm using a UV−vis spectrophotometer (Shimadzu UV-2550). 2.4. Antioxidant Activity and Polyphenol Content. An oxygen-radical absorption capacity (ORAC) assay was used to obtain substantial information regarding the antioxidant capacities of pure quercetin, processed quercetin, and quercetin-loaded Pluronic F127 particles. This assay expresses the results in terms of ORAC units or Trolox equivalents (TE), which is the number of micromoles of Trolox that would have the same antioxidant activity as 1 g of the tested solution. Assays were carried out using a FLUOstar Optima spectrofluorometric system (BMG Labtech, Offenburg, Germany), and the analysis is based on Application Note 140 of BMG Labtech. Total polyphenol contact was determined using the colorimetric method developed by Singleton and Rossi.28 One milligram of sample was diluted in 3 mL of deionized water, and 200 μL of Folin-Ciocalteu reagent (analytical grade, Sigma-Aldrich) was added. The mixture was maintained for 5 min at a temperature at 40 °C under stirring. Afterward, 600 μL of a 20% (w/w) aqueous solution of Na2CO3 was added, and the mixture was maintained at 40 °C under stirring for 30 min. Finally, samples were analyzed by spectrophotometry (FLUOstar Optima, BMG Labtech, Offenburg, Germany) at a wavelength of 765 nm. Gallic acid solutions with concentrations ranging from 0 to 900 ppm, processed in the same way as the samples, were prepared as standards to obtain the calibration line, and the results are expressed as equivalent grams of gallic acid per gram of sample. 2.5. In Vitro Release Tests. In vitro release tests were performed in simulated gastric and intestinal fluids. Simulated gastrointestinal fluids were prepared according to the following procedures: in the case of gastric fluid, 2.0 g of NaCl was dissolved in 1 L of deionized water, and the pH was adjusted to 1.2 by adding HCl. In the case of intestinal fluid, 6.8 g of potassium phosphate was dissolved in 1 L of deionized water, and the pH was adjusted to 6.8 by adding NaOH. Particles of pure quercetin or quercetin/Pluronic coprecipitates were added to the simulated fluids, and the suspensions were maintained at 37 °C and stirred (100 rpm). After predefined periods of 5, 15, 30, 45, 60, 90, 120, and 180 min, samples were taken, and the removed fluid was replaced by fresh simulated gastric or intestinal fluid. Samples were filtered using polytetrafluoroethylene (PTFE) syringe filters with a pore size of 0.45 μm to remove undissolved particles. Afterward, samples were diluted in a mixture of simulated fluid/acetone in

The aim of this study was to study the encapsulation of quercetin in Pluronic F127 by the SAS coprecipitation technique to develop a formulation that can potentially increase the water solubility and bioavailability of quercetin through the formation of micelles in aqueous environments. A detailed morphological and structural characterization of the product is presented herein.

2. EXPERIMENTAL SECTION 2.1. Materials. Quercetin hydrate with a minimum purity of 95% was purchased from Sigma-Aldrich. Ethylene oxide− propylene oxide block copolymer Pluronic F127 [average molecular weight 12600 g/mol, hydrophilic−lipophilic balance (HLB) 22] was provided by BASF (Ludwigshafen am Rhein, Germany). Acetone with a minimum purity 99.5% was provided by Panreac. Fluorescein sodium, Trolox (6-hydroxy2,5,7,8-tetramethylchroman-2-carboxylic acid), and AAPH [2,2′-azobis(2-methylpropionamidine) dihydrochloride] were purchased from Sigma-Aldrich. Phosphate buffer solution (PBS) at pH 7.4 was prepared using NaH2PO4·2H2O and Na2HPO4·12H2O provided by Cofarcas (Burgos, Spain). 2.2. Particle Formation Experiments. The supercritical antisolvent (SAS) precipitation technique was used to produce pure quercetin and quercetin/Pluronic F127 microparticles. Acetone was used as the organic solvent. In this work, a semicontinuous SAS process was used. The pressure and temperature operating conditions, 10 MPa and 313 K, were chosen to operate in the single-phase region of the solvent/CO2 system.27 The solution and antisolvent flow rates were 2 mL/min and 2 kg/h, respectively. Different solution concentrations (ranging from 0.005 to 0.02 g of quercetin/mL acetone) and quercetin/carrier mass ratios (ranging from 2:1 to 1:9) were tested to optimize the particle production process. Figure 1 shows a schematic diagram of the SAS apparatus used in this work. CO2 was taken from a gas cylinder and cooled using ethylene glycol as a refrigerant to maintain it in the liquid state, before it was compressed with a diaphragm pump (model 140.S, DOSAPRO MILTON ROY, Toledo, Spain) and preheated to the desired operating temperature. On the other hand, the liquid solution of quercetin and Pluronic in acetone was pumped with a chromatographic pump (model PU-2080, Jasco, Italy). Both solutions were continuously injected into the precipitation vessel though a concentric tube nozzle, consisting of an inner 1/16-in. tube used to inject the solution and an outer 1/4-in. tube used to introduce CO2. The precipitator was a jacketed vessel with an internal volume of 2.5 L. The formed particles were collected in a frit at the bottom of the precipitator vessel, which consisted of a metallic porous disk used to support a polymeric filter with a pore size of 0.1 μm. Afterward, the effluent from the precipitator was depressurized using a back-pressure valve (model BP66, GO, Spartanburg, SC), the organic solvent condensed after depressurization was collected in a flash vessel, and the gaseous CO2 was vented. Each SAS experiment consisted of three steps. First, the supercritical fluid was introduced into the precipitation vessel until the desired pressure and temperature conditions were reached, and these conditions were maintained constant. Then, the solution was injected at the desired flow rate until at least 100 mL of solution had been processed. Finally, the supercritical fluid flow was maintained constant for at least 30 min to eliminate the remaining organic solvent from the particles. After these three steps, the precipitator was 4320



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a significant reduction in particle size was observed as the concentration of quercetin in the initial acetone solution was reduced. At the lowest concentration of 0.005 g/mL, submicrometric or nanometric quercetin particles, highly agglomerated in flocks of about 1 μm, were obtained. This result indicates that the size and morphology of the particles largely depended on the concentration of the initial solution, with smaller particles being obtained when a lower concentration of quercetin was available for the agglomeration and growth of particles. 3.2. Structural Characterization of SAS-Processed Pure Quercetin. Figure 3 presents differential scanning calorimetry (DSC) thermograms of unprocessed and SASprocessed quercetin. The DSC curve obtained for raw quercetin (Figure 3a) presents two peaks: The first, with an onset temperature of 146 °C, corresponds to the loss of bound water, and the second, with an onset temperature of 319 °C, is related to the melting point of quercetin. A heat of fusion of 163 J/g was measured, in good agreement with the literature results,29 indicating a high crystallinity of the compound. The DSC thermogram of SAS-processed pure quercetin (Figure 3b) also shows a well-defined melting peak. The measured heat of fusion was 152 J/g, very similar to that of unprocessed quercetin. This suggests a high crystallinity of the SASprocessed material, similar to that of the unprocessed compound. However, in DSC analysis of this sample, the peak corresponding to water loss did not appear, indicating that water was removed by CO2 processing, thus producing particles of anhydrous quercetin. This result indicates that SAS precipitation is a suitable technique for the production of anhydrous quercetin, which has a higher commercial value than quercetin hydrate because of its higher biological and antioxidant activity per unit mass of compound. Alessi et al.26

a volume ratio of 1:1, to avoid the possible precipitation of solubilized quercetin as a result of a reduction in temperature or variation in pH during analyses. The concentrations of quercetin in the samples were measured by the UV method previously described in section 2.3.

3. RESULTS AND DISCUSSION 3.1. Micronization of Pure Quercetin. Preliminary experiments on the micronization of pure quercetin by the supercritical antisolvent technique were performed to characterize the crystallization behavior of this compound. Experiments were carried out at a constant temperature and pressure of 40 °C and 100 bar, respectively. As presented in Table 1, different initial concentrations of quercetin in the acetone solution were tested, ranging from 0.02 to 0.005 g/mL. Table 1. Summary of Quercetin SAS Micronization Experiments experiment

quercetin concentration (g/mL)

morphology

QUER-1 QUER-2 QUER-3

0.005 0.01 0.02

small particles small particles fibrous, small particles

Figure 2 presents SEM micrographs of unprocessed quercetin and particles obtained in SAS experiments. To make comparisons easier, all micrographs are presented with the same magnification ratio (5000×, scale bar = 5 μm). As shown in Figure 2a, unprocessed quercetin presented a prismatic particle morphology, with a width of about 2 μm and a length of 20 μm. As shown in Figure 2b−d, upon SAS processing, the prismatic morphology of particles was preserved, but particle size was dramatically reduced. Moreover,

Figure 2. SEM micrographs of particles obtained by SAS micronization of pure quercetin: (a) raw quercetin without processing and (b−d) SASprocessed quercetin with initial concentrations in acetone solution of (b) 0.02, (c) 0.01, and (d) 0.005 g/mL. All micrographs are presented with the same magnification ratio (5000×) and a scale bar of 5 μm. 4321



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This result was further confirmed by FTIR assays, presented in Figure 5. The FTIR spectrum of unprocessed quercetin

Figure 3. Differential scanning calorimetry (DSC) thermograms of (a) unprocessed quercetin, (b) SAS-processed quercetin, (c) physical mixture of quercetin and Pluronic F127 (mass ratio = 1:1), (d) unprocessed Pluronic F127, and (e) SAS-processed Pluronic F127. For clarity, all thermograms are vertically displaced by arbitrary amounts.

Figure 5. FTIR spectra of (a) unprocessed quercetin, (b) SASprocessed quercetin, (c) unprocessed quercetin recrystallized from acetone, (d) SAS-processed quercetin recrystallized from acetone. For clarity, results are vertically displaced by arbitrary amounts.

also observed the dehydration of quercetin upon SAS processing. Figure 4 presents X-ray diffractograms of unprocessed and SAS-processed quercetin. The diffractogram of unprocessed

hydrate agrees well with the literature data.21,25 The broad band at 3500−3000 cm−1 is assigned to a free OH bond vibration, bands at 1660 and 1600 cm−1 are assigned to the stretching vibration of the CO group, the band at 1515 cm−1 is assigned to aromatic groups, the bands at 1310 and 1160 cm−1 are assigned to the COC vibration, and the band at 1010 cm−1 is assigned to aromatic CH groups.31 As shown in Figure 5, the FTIR spectra of unprocessed and SAS-processed quercetin exhibit some significant differences. First, in the spectrum of the SAS-processed material, the OH vibration band at 3500−3000 cm−1 shows a considerable attenuation, which is in agreement with the dehydration of the material by SAS processing observed by DSC analysis (Figure 3). Second, a strong band at 1740 cm−1 appears in the spectrum of SAS-processed quercetin. This band might also correspond to the stretching of CO bonds.32 Several other minor differences in the intensities of different bands can be observed between the spectra of unprocessed and SAS-processed quercetin. The differences in the FTIR spectra can be correlated with variations in the crystalline structure of the material due to the formation of anhydrous quercetin, also observed in DSC and XRD assays. To rule out variations in the chemical structure as possible causes for the differences observed in the FTIR spectra, unprocessed and SAS-processed quercetin particles were recrystallized by evaporation from acetone solutions. As shown in Figure 5, the FTIR spectra of the two recrystallized samples were nearly identical and equivalent to the spectrum of unprocessed quercetin, thus demonstrating that no degradation or variation of the chemical composition of the material occurred upon SAS processing. 3.3. Coprecipitation of Quercetin and Pluronic F127. Table 2 presents a summary of the coprecipitation experiments carried out with quercetin and Pluronic F127. All experiments were performed at constant-temperature and -pressure conditions of 40 °C and 100 bar. As presented in Table 2, several experiments were performed with different concentrations of Pluronic F127, and consequently different mass ratios, in the initial solution, ranging from 2:1 to 1:9 (in units of grams of quercetin/grams of Pluronic). The active compound/

Figure 4. X-ray diffractograms of (a) unprocessed quercetin, (b) SASprocessed quercetin, (c) physical mixture of quercetin and Pluronic F127 (mass ratio = 1:1), (d) SAS-processed mixture of quercetin and Pluronic F127 (mass ratio = 2:1), (e) SAS-processed mixture of quercetin and Pluronic F127 (mass ratio = 1:1), and (f) unprocessed Pluronic F127. For clarity, all diffractograms are vertically displaced by arbitrary amounts.

quercetin displays numerous distinct peaks located at 2θ = 10.8°, 12.5°, 15.8°, 16.2°, 23.8°, 24.5°, 26.7°, and 27.2°, indicating that the compound had high crystallinity. Similar Xray diffractograms of quercetin have been reported by other researchers. 29,30 In the diffractogram of SAS-processed quercetin, several of these peaks show a reduced intensity or are absent, but different well-defined peaks can still be observed. This result indicates that the SAS-processed quercetin was crystalline in nature but had a lower crystallinity than the raw quercetin powder. Notably, a switch between the intensities of the peaks located at 2θ = 26.7° and 27.2° was observed between raw and SAS-processed quercetin samples. The exact reason for this shift is not known, but it might be due to a change to an anhydrous crystalline form. 4322



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micrographs of the particles obtained in several of these experiments. As shown in Table 2, an experiment was carried out studying the micronization of pure Pluronic F127. As presented in Figure 6a, the micronization of this compound by the SAS technique was not successful, given that large, irregular flat particles of more than 200 μm were obtained. This morphology suggests that the particles were formed not by nucleation from the acetone solution by the antisolvent effect, but rather by solidification from a polymer melt. Indeed, in a previous work, it was shown that CO2 interacts strongly with Pluronic F127, leading to a reduction in the melting temperature of this compound of over 15 °C at moderate pressures until temperatures near the 40 °C employed in SAS experiments.34 Experimental results regarding the phase behavior of similar systems composed of organic solvent + supercritical CO2 + low-melting-temperature polymer, such as dichloromethane + CO2 + poly(ethylene glycol),35 show that, in these ternary systems, the melting-temperature-reduction effect of pure CO2 can be increased by a liquid−liquid phase split induced by the partition of the organic solvent between the supercritical phase and a polymer-rich phase. However, detailed phase equilibrium data demonstrating the formation of a liquid−liquid phase split is not available for the specific system considered in this work (acetone + CO2 + Pluronic F127 at 40 °C). Although operation near the melting region is disadvantageous for the micronization of a pure polymer, it is a favorable condition for coprecipitation experiments, because it can facilitate the

Table 2. Summary of Quercetin + Pluronic F127 SAS Coprecipitation Experiments experiment

mass ratio (g of quercetin/g of F127)

quercetin concentration (g/mL)

morphology

F127-1

−

−

QUER +F127-1 QUER +F127-2 QUER +F127-3 QUER +F127-4 QUER +F127-5 QUER +F127-6 QUER +F127-7 QUER +F127-8

2:1

0.02

1:1

0.02

large flat particles small particles with fibers small particles

1:1.5

0.02

small particles

1:1.7

0.02

small particles

1:2

0.02

plastification

1:9

0.02

plastification

1:2

0.01

small particles

1:9

0.01

plastification

polymer mass ratio was chosen as the main process parameter to be analyzed because previous results on the encapsulation of active compounds by copreciptiation with polymers using the SAS technique indicated that this ratio is the main parameter influencing particle morphology.33 Figure 6 shows SEM

Figure 6. SEM micrographs of particles obtained by SAS coprecipitation of Pluronic F127 + quercetin: (a) pure F127, (b) quercetin + Pluronic F127 in a mass ratio of 2:1 (experiment QUER+F127-1 of Table 2), (c) quercetin + Pluronic F127 in a mass ratio of 1:1 (experiment QUER+F127-2 of Table 2), (d) quercetin + Pluronic F127 in a mass ratio of 1:1.7 (experiment QUER+F127-4 of Table 2). Micrographs b−d are presented with a magnification ratio of 5000× and a scale bar of 5 μm, whereas micrograph a is presented with a magnification ratio of 100× and a scale bar of 200 μm. 4323



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encapsulation of the active compound through the formation of a polymer film over active-component particles, avoiding the crystallization of segregated particles of active compound and polymer. Indeed, as shown in Figure 6b−d, coprecipitation experiments of quercetin with Pluronic F127 yielded a completely different morphology than experiments with pure polymer. The morphologies obtained indicate that querectin particles acted as nucleation sites for the formation of a polymer film and that this film of polymer restrained the growth of quercetin particles. Moreover, important variations in the morphology of the particles were observed as the quercetin/polymer mass ratio was reduced. At the highest mass ratio tested (2:1, experiment QUER+F127-1, Figure 6b) spherical particles, probably consisting of quercetin encapsulated in Pluronic F127, were observed, together with prismatic particles with the same morphology as obtained in experiments with pure quercetin (Figure 2b), which were probably crystals of this compound not encapsulated in the polymer. When the quercetin/polymer mass ratio was reduced, these segregated crystals were not formed. Experiments with a quercetin/polymer mass ratio of 1:1 yielded a highly homogeneous product consisting of spherical particles of about 1 μm (Figure 6c). However, when the quercetin/polymer mass ratio was further reduced, the product showed more agglomeration, with larger particles of polymer fused together as in Figure 6d. Experiments with quercetin/polymer mass ratios below 1:2 were not successful, because a plasticized product was obtained, as in the experiment with pure Pluronic F127 described earlier. As shown by the results of experiment QUER+F127-7 in Table 2, by reducing the total concentration of both quercetin and Pluronic, a successful coprecipitation was also achieved at a quercetin/polymer mass ratio of 1:2. This might be a consequence of the reduction of the size of quercetin particles, discussed in section 3.1. However, experiments with this reduced concentration of quercetin and lower quercetin/ polymer mass ratios again rendered plasticized products. Thus, the experimental results indicate that viable conditions for SAS coprecipitation of quercetin with Pluronic F127 are limited to a narrow range of quercetin/polymer mass ratios, from 1:1 to 1:2, in which quercetin particles can act as nucleation sites for the formation of polymer films and the concentration of polymer is high enough for the complete encapsulation of quercetin particles. In comparison, in a previous study of the coprecipitation of β-carotene in poly(ethylene glycol) (PEG) polymers, much higher polymer/active compound mass ratios could be employed (up to 20 g of carrier per gram of active compound33), but the operating temperatures employed (down to 15 °C) were far below the melting temperature of PEG. Therefore, the narrowing of the operating region is due to operation near the melting point of the polymer, which leads to the formation of fused polymer films if the polymer/active compound mass ratio is too high. Table 3 presents the compositions of the quercetin/Pluronic coprecipitates measured by UV spectroscopy, as described in section 2.3, compared with the theoretical compositions calculated according to the amounts of active compound and carrier in the initial solution. As presented in this table, very good agreement between the experimental and theoretical compositions was obtained, indicating a high yield of precipitation of both compounds. 3.4. Structural Characterization of SAS-Processed Quercetin−Pluronic F127 Coprecipitates. The DSC

Table 3. Experimental and Theoretical Concentrations of Quercetin in Coprecipitation Experiments with Pluronic F127 experiment QUER +F127-1 QUER +F127-2 QUER +F127-3 QUER +F127-4 QUER +F127-7

theoretical quercetin loading (wt %)

experimental quercetin loading (wt %)

66

56 ± 1.1

50

50 ± 1.5

40

42 ± 1.7

37

40 ± 4

33

35 ± 1.8

thermogram of unprocessed Pluronic presented in Figure 3 exhibits a melting peak at 59 °C, characteristic of this compound. In comparison, the corresponding peak of SASprocessed Pluronic is wider, indicating a certain reduction of the crystallinity of this compound upon SAS processing. Figure 7 presents DSC thermograms of different quercetin/ Pluronic samples obtained by SAS processing. In the

Figure 7. DSC thermograms of SAS-processed quercetin and Pluronic F127 at mass ratios of (a) 2:1, (b) 1:1, (c) 1:1.5, (d) 1:2, and (e) 1:9.

experiment performed with the highest quercetin/Pluronic mass ratio (2:1), the melting peak of quercetin was clearly visible. However, as the proportion of polymer in the mixture was increased, the melting peak of quercetin was dramatically reduced and gradually shifted to lower temperatures, and at quercetin/Pluronic mass ratios below 1:2, this melting peak was no longer visible. These results indicate the presence of segregated quercetin crystals at high quercetin/Pluronic mass ratios, whereas experiments at lower mass ratios yielded solid solutions of quercetin and polymer without segregated quercetin crystals. These results are in agreement with the morphology of particles observed by SEM (Figure 6). Moreover, DSC traces of SAS-processed mixtures showed a peak near 180 °C that might correspond to the loss of bound water, indicating that, in these mixtures, quercetin was not as efficiently dehydrated as in experiments with pure compound, probably as a result of the formation of a polymer film over the quercetin particles. In the X-ray diffractograms presented in Figure 4, two welldefined peaks located at 2θ = 19° and 23° can be observed in the assays with Pluronic, in agreement with literature data.30 As expected, the diffractogram of a physical mixture of quercetin 4324



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attributed to quercetin at 3500−3000 cm−1 (OH vibration) and 1660 and 1600 cm−1 (CO). As in experiments with pure quercetin, in SAS-processed mixtures, the OH band appeared attenuated because of the dehydration of the compound, and an additional peak at 1740 cm−1 was observed. Moreover, the results showed a clear dependence of the relative intensity of the peaks corresponding to Pluronic on the concentration of Pluronic in the mixture. Apart from this, no other variations in the FTIR spectra of coprecipitates, such as shifts of characteristic bands or intensification/attenuation of these bands, were observed, indicating that complexation or chemical association between the carrier and quercetin did not occur. 3.5. Evaluation of Antioxidant Activity. Table 4 presents the results obtained by ORAC antioxidant activity tests. As reported in this table, SAS-processed quercetin showed a significantly higher antioxidant activity than unprocessed quercetin. To a certain extent, this might be due to the dehydration of the compound. Indeed, hydration water accounts for approximately 10% of the weight of unprocessed quercetin, so removing water from the unprocessed compound would increase its antioxidant actitivity from 6300 μmol of TE/ g of product to approximately 7100 μmol of TE/g of product. The additional increase in the antioxidant activity might be due to a purification of the product through the removal of lowmolecular-weight degradation compounds that can remain solubilized in supercritical CO2 during SAS processing. A similar purification effect was observed in the SAS processing of lutein, a carotenoid antioxidant.36 On the other hand, all SAS-processed coprecipitates of quercetin and Pluronic exhibited equivalent antioxidant activities per unit mass of quercetin in the product, equal to that of pure, SAS-processed quercetin within experimental error. Furthermore, the results presented in Table 4 indicate that the antioxidant activities of the samples were entirely preserved after 45 days of storage. The results of the antioxidant activity tests were confirmed by total polyphenol measurements, reported in Table 5. As presented in this table, all SAS-processed samples exhibited equivalent results that were slightly higher than the results for unprocessed quercetin. 3.6. In Vitro Release Tests. Figures 9 and 10 show the results of release tests in simulated gastric and intestinal fluids , respectively. As presented in Figure 9, SAS-processed pure quercetin exhibited faster dissolution in gastric media than the unprocessed product, as well as a higher final solubility. This result can be attributed to the reduction of the particle size of the material achieved by SAS micronization, which increased the available surface area and improved the dissolution, and the reduction of the crystallinity, which reduced the stability of the

and Pluronic was found to reflect a simple superposition of the diffractograms of the two pure compounds (Figure 4c). In contrast, in SAS-processed samples, characteristic peaks of quercetin were attenuated, and peaks of Pluronic were not observed (Figure 4d,e). This result indicates that, compared with particles obtained by SAS processing of pure quercetin, in coprecipitation experiments, particles of quercetin had a lower crystallinity or formed a molecular dispersion in a matrix of amorphous polymer. The attenuation of the peaks was more pronounced in the experiments at lower quercetin/Pluronic mass ratios, in agreement with the SEM results indicating that experiments with larger proportions of polymer yielded more homogeneous products without segregated quercetin crystals (Figure 6). As previously discussed in the Introduction, some encapsulation techniques and carrier materials can be used to produce complexes with chemical association between active and carrier materials, such as inclusion complexes or solid micellar structures,7,21 that can substantially enhance the aqueous solubility and dissolution rate of the active compound, as, in these cases, the inclusion complexes or micellar aggregates would already be formed in the solid phase before dissolution in water. The possible formation of such interaction bonds can be detected by FTIR spectroscopy.8 Figure 8

Figure 8. FTIR spectra of (a) Pluronic F127, (b) physical mixture of quercetin and Pluronic F127 (1:1), (c) quercetin/Pluronic F127(1:2), (d) quercetin/Pluronic F127(1:1), (e) quercetin/Pluronic F127(2:1). For clarity, all spectra are vertically displaced by arbitrary amounts.

presents FTIR spectra of quercetin/Pluronic samples. The spectrum of a physical mixture of the two compounds displayed a combination of the peaks of the two compounds, including characteristic peaks of Pluronic F127 at 2870 cm−1 (aliphatic CH stretch) and 1100 cm−1 (COC stretch) and peaks

Table 4. Results of ORAC Antioxidant Activity Tests Presented as Antioxidant Activity per Unit Mass of Total Product (Quercetin + Polymer) and per Unit Mass of Quercetin in the Product antioxidant activity (μmol of TE/g of product)

antioxidant activity (μmol of TE/g of quercetin)

sample

composition (wt % quercetin)

initial

after 45 days

initial

after 45 days

unprocessed quercetin SAS-processed quercetin physical mixture QUER+F127-1 QUER+F127-2

100 100 50 58 52

6300 ± 300 10900 ± 500 2500 ± 400 6700 ± 700 6500 ± 800

6300 ± 300 10900 ± 500 NA 7000 ± 900 6000 ± 1000

6300 ± 300 10900 ± 500 5200 ± 400 11600 ± 700 12500 ± 800

6300 ± 300 10900 ± 500 NA 12000 ± 900 11000 ± 1000

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and 2.5 ppm in the case of unprocessed quercetin. This effect is related to the reduction of the particle size and crystallinity of quercetin, which, as previously described, displaced the phase equilibrium toward the solution and micellar phases. It is also related to the production of a homogeneous solid solution of quercetin in the amorphous polymer matrix, as observed in structural characterization assays described in section 3.4, which eliminated kinetic barriers for the dissolution of the compounds and the formation of quercetin-loaded micelles, thus increasing the dissolution rate. The results of release experiments in simulated intestinal fluid are presented in Figure 10. In general, a higher solubility of quercetin was observed in experiments in simulated intestinal fluid than in experiments in simulated gastric fluid, but the general trends of variation between unprocessed, SASprocessed, and encapsulated quercetin were the same in both cases. As presented in this figure, in this case, a significant difference was again observed between the results with unprocessed and SAS-processed pure quercetin in both the dissolution rate and the final solubility. Similarly, SASprocessed mixtures of quercetin and Pluronic showed a faster solubilization and a higher final solubility than a simple physical mixture of these two compounds. This result indicates that the properties of the product obtained by SAS processing described in previous sections, including the reduction of particle size and the production of a homogeneous dispersion of the active compound in an amorphous polymer matrix, have a positive impact on the dissolution behavior of the formulation.

Table 5. Total Polyphenol Content of Unprocessed Quercetin and SAS-Processed Samples g of gallic acid equivalents/g of quercetin raw quercetin SAS-processed quercetin QUER+F127-2 (1:1) QUER+F127-1 (2:1) QUER+F127-3 (1:1.5)

1.31 1.49 1.32 1.52 1.47

Figure 9. Drug release tests in simulated gastric fluid.

4. CONCLUSIONS Quercetin and quercetin/Pluronic F127 particles were successfully coprecipitated from acetone solutions by a semicontinuous SAS process. As indicated by the results of morphological and structural assays, a significant reduction in particle size and a homogeneous dispersion of quercetin in an amorphous polymer matrix were obtained with this technique. Degradation of the product was not observed, and SASprocessed compounds showed higher antioxidant activities than unprocessed compounds because of the elimination of hydration water and purification of the compound. Because of the morphological and structural properties conferred by SAS processing, formulations exhibited faster dissolution and a higher final solubility in simulated gastric and intestinal fluids than unprocessed quercetin or a physical mixture of quercetin with Pluronic.

Figure 10. Drug release tests in simulated intestinal fluid.

particles and therefore increased their solubility in the simulated gastrointestinal fluids. On the other hand, samples formulated by a simple physical homogenization of quercetin with Pluronic exhibited faster dissolution and a higher solubility than samples of pure quercetin. The increase in the solubility of quercetin can be related to the capacity of Pluronic to form micelles in aqueous media that can host the active compound in their inner cavities, thus increasing its solubility. This process is spontaneous and driven by phase equilibrium partitioning of quercetin among the solid phase, the aqueous solution, and the micellar phase, and it does not require any specific microstructure of the particles, a behavior frequently observed with many similar micelle-forming surfactant carriers.37,38 However, SAS-processed coprecipitates allowed for a 4-fold increase of the solubility with respect to that of a simple physical mixture of the compounds, reaching a final solubility of 16 ppm, compared to 3.5 ppm in the case of physical mixtures

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +34 983184077. E-mail: [email protected]. Author Contributions

M.F. performed SAS coprecipitation experiments and product characterization assays. R.B. collaborated in SAS coprecipitation experiments and performed in vitro release tests. B.G. performed preliminary experiments on the recrystallization of pure quercetin. Á .M. and M.J.C. planned the experimental program and supervised the research. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest. 4326



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ACKNOWLEDGMENTS This research was partially funded by the Spanish Ministry of Economy and Competitiveness through Project ENE201124547. Á .M. thanks the Spanish Ministry of Economy and Competitiveness for a Ramón y Cajal Research Fellowship. R. B. thanks the Conselho Nacional de Desenvolvimiento Cientifico e Tecnológico and CAPES of Brazil for a mobility grant.

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